new york universitysun-lab.med.nyu.edu/files/sun-lab/attachments/cpcb.ch09...general characteristics...

CHAPTER 9Cell Adhesion

INTRODUCTION

Cell-adhesive interactions determine the organization of tissues and mediate and guideprecise cell migrations during embryonic development, inflammation, the immune

response, and wound repair. They also help to regulate gene expression, growth, differ-entiation, and apoptosis. Research into cell adhesion has undergone dramatic evolutionover the past two to three decades, from phenomenological studies of the biophysical andmorphological mechanisms used by cells to adhere to other cells and the extracellularmatrix, to the discovery and in-depth mechanistic analyses of a variety of specific celladhesion systems, and then to the recent paradigm shift whereby adhesion molecules arenow viewed as activators or regulators of a remarkably wide range of signal transductionpathways.

FUNCTIONS OF ADHESION MOLECULES

Cell adhesion molecules function by forming specific protein-protein or protein-carbo-hydrate bonds at the cell surface to mediate cell interactions. In addition, cell adhesionmolecules or cell adhesion receptors often form direct links to multimolecular proteincomplexes on the cytoplasmic face of the plasma membrane. These cytoplasmic adhesionand signaling complexes interact with the cytoskeleton and signal transduction pathways.As a result, cell adhesion not only links cells with other cells and the extracellular matrix,but also helps to integrate extracellular physical information with the major signaltransduction pathways within cells.

Cell adhesion is often categorized into cell-to-cell adhesion and cell-to-substrate adhe-sion. In each case, the physical structures that mediate cell adhesion can be eitherspecialized adhesive structures or broad expanses of plasma membrane. For example,cells can initially adhere to other cells along large areas of plasma membrane usinggeneral-purpose adhesive molecules such as cadherins. But they can also form attach-ments to each other by using specialized adhesive structures, such as desmosomes,adherens junctions, and tight junctions. Each type of junctional complex involves specificadhesion-molecule components, such as cadherins and associated cytoplasmic compo-nents involved in linkage to the actin-containing cytoskeleton. Fibroblasts can also formadhesions to other fibroblasts, but they most characteristically form adhesive interactionswith extracellular matrix molecules. Such cell-to-matrix adhesion sites can also be eitherbroad zones or specialized structures. For example, rapidly migrating cells typicallyinteract with tissue culture substrates through broad areas of contact termed “closecontacts,” and the entire basal surface of epithelial cells adheres via integrins to theirunderlying basement membranes. Specialized adhesive structures include the hemides-mosomes of certain epithelial cells and the focal adhesions of fibroblastic and endothelialcells. The protein composition of the complexes involved in cell-to-matrix adhesions cansometimes involve a few of the same proteins as in cell-to-cell adhesions, such as vinculinand actin, but they mainly consist of distinct types of adhesive, cytoskeletal, and signaltransduction molecules.

Supplement 18

Contributed by Kenneth M. YamadaCurrent Protocols in Cell Biology (2003) 9.0.1-9.0.9Copyright © 2003 by John Wiley & Sons, Inc.

9.0.1

Cell Adhesion

GENERAL CHARACTERISTICS OF CELL ADHESION PROTEINS

Cell adhesiveness generally involves specific binding of a cell adhesion protein or receptorto a target molecule. Adhesion mediated by the binding of an adhesion molecule to thesame type of protein on a neighboring cell is termed a “homophilic” interaction. Membersof the calcium-dependent cadherin family are major mediators of such interactions, and somecalcium-independent adhesion proteins, such as N-CAM, can also provide homophilicinteractions. Cadherin adhesion molecules characteristically form tightly packed dimeric andmultimeric complexes that mediate adhesion with high avidity via cooperativity, even thoughthe affinity of a single cadherin protein-protein interaction alone is not high.

A particularly common type of adhesive interaction involves the binding of a receptor toa specific ligand. In the process of cell-to-cell adhesion, the target protein of an adhesionreceptor can be either a “counter-receptor” or a complex carbohydrate linked to a proteinanchor in the plasma membrane. In cell-to-matrix interactions, a plasma membraneadhesion receptor such as an integrin binds to an adhesive extracellular matrix protein.For example, a number of types of integrin receptors can bind to fibronectin, laminin, orcollagen. Integrin receptors directly mediate cell adhesion, migration, and anchorage tothese structural components of the extracellular matrix.

Molecules that mediate cell-cell or cell-matrix adhesion fall into two broad structuralclasses. One type is anchored to the plasma membrane, often as a transmembrane protein.These membrane-anchored molecules are generally receptors, homophilic adhesionmolecules, or counter-receptors. They often consist of an extracellular domain containingone or more specific cell interaction domains or sites, as well as a stalk region, ahydrophobic transmembrane domain, and a cytoplasmic domain or tail. This type ofadhesion molecule is often likely to be involved in the transmembrane transmission ofsignals after binding to their target molecule.

The second broad class of adhesion molecules consists of cell surface or extracellularmatrix proteins (see Chapter 10) that contain domains involved in cellular adhesion.Nearly all matrix proteins contain such sites. For example, this class includes fibronectin,laminins, vitronectin, collagens, and many other extracellular proteins. These proteinscontain one or more cell-binding domains, which are comprised of a primary recognitionmotif consisting of a short peptide sequence (e.g., Arg-Gly-Asp or Leu-Asp-Val) and oftena synergy site or other structural feature that substantially enhances receptor-bindingspecificity and affinity.

Adhesion molecules frequently have many of the following characteristics (for reviews,see Alberts et al., 1994; Chothia and Jones, 1997; Edelman and Thiery, 1985; Gumbiner,1996; Hay, 1991; Richardson and Steiner, 1995).

1. The backbone structure of adhesion proteins is often based on multiple repeats ofprotein motifs, such as the immunoglobulin (Ig) motif, EGF repeat, or fibronectinmotif. The basic immunoglobulin repeat is quite common, as is the structurally relatedfibronectin type III repeat.

2. They have specialized functional domains, including one or more domains for bindingmolecules on other cells or in extracellular matrix. Often a domain for forming dimersor higher polymers is also present, as well as another for binding to complexcarbohydrates.

3. They often bind with only moderate affinity, e.g., in the range of Kd = 10−6 to 10−7 Mfor fibronectin, and as low as 10−4 M for leukocyte adhesion molecules involved inrolling adhesion. This modest affinity appears to be important for allowing dynamicchanges in cell adhesions and to permit cell migration involving cyclic attachmentand detachment of cell adhesion sites to a substrate or other cells.

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9.0.2

Introduction

4. They often function by clustering to generate complexes of high avidity: adhesionmolecules such as integrins and cadherins often organize into large local clusters oraggregates in the plane of the plasma membrane, which can produce strong totalavidity as a result of the summation of the otherwise weak binding of many individualmolecules.

5. Their binding functions can be regulated by activation, e.g., “inside-out” signalingthat changes their ability to bind ligands effectively; examples are integrin activationduring leukocyte adhesion and platelet activation, and the suppression of the cadherinsystem by tyrosine phosphorylation.

MAJOR FAMILIES OF CELL ADHESION MOLECULES

Adhesion molecules that are anchored in the plasma membrane include several largegroups of proteins that share common structural motifs, such as the immunoglobulinrepeat. Cadherins comprise a large family of proteins particularly centrally involved incell-to-cell adhesive interactions (Takeichi, 1990; Yap et al., 1997; see UNIT 9.3). Cadherinson one cell bind in homophilic fashion to the same type of cadherin on other cells bymeans of specific cell interaction domains, which can include the short peptide recogni-tion sequence His-Ala-Val. The cytoplasmic domain of cadherin molecules charac-teristically binds to catenins, which provide direct linkages to the actin cytoskeleton (e.g.,via α- and β-catenins).

The “classical” cadherins such as E-cadherin and N-cadherin mediate adhesion over broadexpanses of cell-cell contact, or they can become further organized into adherens-typejunctions that are linked to cytoplasmic molecules such as vinculin and actin. A numberof other types of cadherins have been discovered recently, whose functions are probablyalso adhesive, but which remain to be characterized. Finally, certain highly specializedcadherins found exclusively in desmosomes, termed desmocollins and desmogleins, linkcells together at particularly strong attachment sites connected to intermediate filamentssuch as keratins or vimentin.

Cadherin activity is quite sensitive to calcium ion concentration, which accounts for theability of calcium chelators such as EDTA to dissociate tissues into their individualcomponent cells. Chelation of divalent cations appears to disrupt conformation, so thatthe cadherin molecules become quite sensitive to general proteolytic attack; this latterproperty has been used to classify cell-cell adhesion molecules (see UNIT 9.3). Althoughdesmosomal and other cadherins can be found widely in epithelia of organisms at all ages,the cadherins are particularly important during embryonic development, when they appearto organize groups of cells and tissues by specific cell-cell adhesions. They help to definetissues by binding primarily to cadherins of the same type, rather than to other cadherinson unrelated cell types; this activity can lead to “sorting out” of different types of cellsfrom others.

Integrins are nearly ubiquitous cell surface receptors for a wide variety of extracellularmatrix proteins, as well as for “counter-receptor” ligands on other cells. There are morethan 20 distinct integrin subunits, which are combined to form heterodimers that alwaysconsist of one α and one β subunit. Genetic loss of almost any integrin subunit leads todisease or death, often during embryonic development or near the time of birth. Integrinmolecules have a head domain containing a ligand-binding site, two spindly legs, andusually rather short cytoplasmic domains. Integrin binding is often inhibited by depletionof divalent cations.

CAMs (cell adhesion molecules) and integrin counter-receptors are structurally relatedby their use of the immunoglobulin repeat, and they often have the term “-CAM” in theirnames, yet they differ functionally. Molecules such as NCAM are calcium-independent,

Current Protocols in Cell Biology Supplement 18

9.0.3

Cell Adhesion

homophilic adhesive molecules that bind to the same type of molecule on an adjacent cellsurface, often a cell of the same tissue type (Edelman and Crossin, 1991). In contrast,counter-receptors such as the ICAMs and VCAM have specialized peptide recognitionsites that are bound specifically by integrins such as LFA-1 (CD11a/CD18 or αLβ2) orVLA-4 (α4β1), which are often present on other types of cells (e.g., endothelial cells andlymphocytes). The functions and sites of expression of these molecules can consequentlydiffer widely. For example, molecules such as NCAM are implicated in embryonicdevelopmental events such as axonal guidance and bundling, whereas ICAM counter-re-ceptors are present as targets for binding by cells circulating in blood. The levels of thesecounter-receptors on the cell surface can often also be regulated rapidly in response tocytokines, for example during inflammatory responses.

A family of transmembrane proteins termed ADAMs (membrane proteins with a disin-tegrin and metalloprotease domain; Wolfsberg and White, 1996) contain a protease-likedomain plus an integrin recognition site in the disintegrin domain. They appear to playroles in cell adhesion; for example, one member of this family is present on sperm andhas been implicated in sperm-egg adhesion. Syndecans are cell-surface heparan sulfateproteoglycans with a protein core that crosses the plasma membrane and terminates in acytoplasmic tail. Syndecans appear to function as “co-receptors,” mediating signaling inassociation with a primary adhesion molecule. For example, syndecans synergize withfibronectin during formation of focal adhesion sites and form linkages to protein kinaseC (Woods and Couchman, 1998).

Adhesive extracellular matrix molecules are described in Chapter 10. One interestingaspect of their function involves the striking effects of adsorption or attachment to asubstrate. Molecules such as fibronectin and vitronectin circulate in blood at relativelyhigh concentrations (e.g., 100 to 300 µg/ml), yet binding to cell surface receptors isrelatively limited. On the other hand, even very low concentrations of the same proteins(1 to 10 µg/ml) bind to substrates and then mediate strong cell adhesion. This functionalenhancement of binding of cells to molecules attached to a substrate has been variouslyascribed to activation of molecules such as fibronectin and vitronectin by conformationalchanges resulting from binding to a substrate, to enhanced ability to interact with cells dueto multivalency, or to a physical chemical enhancement in free energy (overall avidity) dueto immobilization of a ligand. Whatever the mechanisms, it is clear that the binding ofextracellular adhesion molecules to substrates can enhance functional activity, which appearsto be important biologically to generate strong adhesion only when a circulating ligand isimmobilized. For example, in wounds, immobilized fibronectin forms a crucially importantprovisional matrix that permits adhesion and migration of cells to close the wound.

FUNCTIONS OF CELL ADHESION MOLECULES IN SIGNALTRANSDUCTION AND CYTOSKELETON

The functions of cell adhesion molecules and their receptors can be understood intuitivelyas physical mechanisms by which cells attach to other cells, adhere to extracellular matrixmolecules, and provide traction during cell migration. This concept can be extended tointracellular structural effects—i.e., the organizing of the cytoskeleton. For example,transmembrane adhesion proteins provide membrane anchors for a host of cytoskeletalproteins such as keratin or vimentin in desmosomes and hemidesmosomes, actin micro-filaments and other proteins in epithelial adherens junctions, and a number of adhesionplaque proteins and actin in cell-substrate adhesion plaques (e.g., see Jockusch et al.,1995; Yamada and Geiger, 1997).

It is now clear, however, that adhesion molecules also play crucial roles in helping totrigger or modulate many major signal transduction pathways. In fact, their signaling


9.0.4

Introduction

functions are probably at least as important in cell biology as their attachment andcytoskeletal activities. A notable characteristic of the intracellular complexes of proteinsinduced by binding of adhesion molecules is that their formation also induces accumula-tion of a variety of signal transduction molecules that can trigger downstream signalingpathways (Clark and Brugge, 1995; Schwartz et al., 1995; Lafrenie and Yamada, 1996).As a consequence, adhesion proteins are actually cell-interaction proteins that havemultiple functions in the bidirectional transfer of information at the cell surface. They canmediate outside-in transfers of signaling information, such as cellular responses tobinding of specific extracellular matrix proteins involving the activation of intracellularsignaling, but they can also mediate inside-out information in which intracellular signalsmodulate the activity of integrins and assembly of extracellular matrix. Even classicaladhesive proteins such as fibronectin can trigger a bewildering range of activities afterbinding by integrins; these can include activation or modulation of many well-knownmammalian signal transduction pathways (such as tyrosine phosphorylation, MAP ki-nases, protein kinase C, Ca2+ and H+ fluxes, and phosphoinositide pathways) which canactivate specific gene transcription, mediate anchorage-dependent growth stimulation,and prevent apoptosis.

Two structural regions in transmembrane adhesion molecules that allow them to functionas signal transduction receptors are their extracellular ligand-binding domains and theircytoplasmic domains. The ligand-binding domains are obviously essential for binding toextracellular molecules, but in at least some cases, they have additional, intriguing roles.For integrin adhesion receptors, there appear to be separable functions for ligand occu-pancy (filling the binding site with a ligand), as opposed to receptor clustering (whichcan be induced by multivalent ligands such as fibrils of fibronectin or collagen). Thesetwo inputs can synergize to promote the accumulation of specific cytoskeletal proteinssuch as α-actinin and actin to form strong adhesions (Yamada and Miyamoto, 1995).

Even though they lack intrinsic enzymatic activities, integrins appear to function assignaling receptors and regulators of actin cytoskeletal organization by recruiting othermolecules to bind to their cytoplasmic domains. Integrins appear to bind directly to certaincytoplasmic proteins, such as talin, α-actinin, and tensin, in a process that is sometimesregulated by ligand occupancy on the outside of the cell. Also important, however, aredocking proteins such as focal adhesion kinase, which can bind to integrins as well asbinding to at least eight cytoplasmic molecules to form molecular complexes. Otherdocking proteins such as p130Cas probably increase the repertoire of cross-linked andcomplexed proteins. Integrin clustering appears to play a central role in forming largecomplexes that can consist of over 30 different types of molecules. These integrin-inducedmultimolecular complexes can serve as signaling centers, e.g., for tyrosine phosphoryla-tion and MAP kinase activation (reviewed by Yamada and Miyamoto, 1995; see UNIT 14.2

and UNIT 14.3 for protocols measuring these post-translational modifications).

Cell-to-cell adhesion molecules also appear to undergo a very similar process of localclustering, complex formation, binding of cytoskeletal proteins such as actin, and accu-mulation of signaling molecules (Kirkpatrick and Peifer, 1995; Yap et al., 1997; Yamadaand Geiger, 1997). It will become increasingly important to understand how theseprocesses are controlled. The cadherin system can be suppressed by tyrosine phosphory-lation mediated by v-Src kinase, and certain integrin functions can be down-modulatedby the phosphatase PTEN (Takeda et al., 1995; Tamura et al., 1998). There are likely tobe large numbers of other regulatory pathways that affect the widespread intracellulareffects of cell-cell and cell-substrate adhesion molecules on cytoplasmic processes. It isclear that adhesion molecules are crucial, integral components of the basic signaling andregulatory mechanisms of cells, providing dynamic links to the external environment.


9.0.5

Cell Adhesion

CELL ADHESION ASSAYS

A crucial first step for effective analysis of a cell adhesive process is to characterize it ina quantitative in vitro assay. Cell-to-substrate adhesion assays are used to examine theability of cells to attach to matrix molecules, such as fibronectin or laminin, and to determinewhich specific adhesion receptors are involved using antibodies or synthetic peptide inhibi-tors. Roles of cytoplasmic molecules such as cytoskeletal proteins and signaling moleculescan also be evaluated with these assays using pharmacological inhibitors of intracellularprocesses—for instance inhibitors of kinases and other molecules.

There are several types of cell-substrate adhesion assays, which can quantitate (1) cellattachment, (2) cell spreading, or (3) detachment of previously adherent cells. Each assaymeasures different parameters of the adhesion process: cell attachment assays generallydetermine the numbers of cells that can attach to a substrate in a specific time period afterwashing off nonattached cells (UNIT 9.1); cell spreading assays determine the percentageof cells that show spread morphologies, or measure the surface area of spread cells, aftercertain times of incubation (UNIT 9.1); and cell detachment assays measure the ease withwhich cells can be detached after a particular period of time, for example by determiningthe numbers of cells detached from a dish using each of a series of increasing centrifugalforce levels (UNIT 9.2). Although conceptually distinct, these three types of assays can inpractice have features that overlap significantly.

In order to compare these assays, it is useful to consider the steps in cell-substrate adhesionand spreading. The first step in cellular adhesion to a substrate is the attachment of plasmamembrane receptors or other cell interaction molecules to substrate molecules such asextracellular matrix (ECM) proteins (Chapter 10). For example, if cells are cultured ontissue culture dishes or glass in serum-containing medium, they often adhere to serumproteins such as vitronectin and fibronectin that are nonspecifically adsorbed to thesubstrate. Integrins frequently mediate such adhesions (e.g., attachment of cells to anextracellular matrix protein), although various other cell attachment proteins, such as theselectins, also exist.

The binding of integrins to ligands can occur even at 4°C, because it is a directprotein-protein interaction. However, this mechanism of cell adhesion is initially quiteweak and can often require centrifugation of the cell against the substrate to produce closeenough apposition of the plasma membrane to substrate proteins for effective binding. Incontrast, normal adhesion appears to involve active cellular processes involving theplasma membrane and the cytoskeleton. The latter, poorly understood process of cy-toskeletal strengthening of adhesions can produce enormous increases in attachmentstrength (see UNIT 9.2 for a conceptual discussion). This strengthening phenomenon maybe related to the formation of multimolecular complexes in adhesion structures containinga variety of cytoskeletal and signal transduction proteins (Burridge et al., 1997; Yamadaand Geiger, 1997). Cell attachment assays are generally performed shortly after platingcells before much cell spreading can occur (e.g., within 10 to 20 min for fibroblasts).

Attachment is rapidly followed by spreading of the cell on the substrate in a dynamicadherence response involving cell movements; spreading can become maximal by 30 to60 min. Even in simple attachment assays, it appears likely that there are some contribu-tions from cell spreading, as the area of attachment of the cell to the substrate expands,accompanied by increasing organization of the cytoskeleton. As spreading and firmeradhesion ensue, cells often form strong focal adhesions to the substrate that are sites oftermination of actin microfilament bundles (also known as stress fibers, since the bundlesappear to follow lines of linear stress between focal adhesions). As cells attach and spread,they rapidly acquire resistance to detachment, for example by centrifugal force.

Although assays for cell attachment, spreading, or detachment each focus on one particu-


9.0.6

Introduction

lar facet of cell-substrate adhesion, each assay has strengths, weaknesses, and partialoverlaps with the others. Attachment assays are conceptually the simplest, but nonethelessusually unavoidably involve an element of measuring resistance to detachment of the cellsby shear forces. In order to rinse unattached cells out of 96-well plates, medium must bepoured in and then aspirated or flicked out, thereby generating substantial shear forces asthe meniscus passes over attached cells. Cell spreading assays can provide rapidsemiquantitative data and evaluate the cytoskeletal facet of cell-adhesive interactions. Asa consequence, however, they do not measure only the simple interaction of an adhesionreceptor with its ligand, but instead the more complete adhesion response. Spreadingassays also involve an element of judgment in choosing to record whether a cell is spreador not spread, which can sometimes perturb a researcher unaccustomed to microscopy.The advantage of centrifugal detachment assays is that they can provide direct physicalmeasurements of adhesive strength as measured in force units (× g). However, this sortof assay is particularly sensitive to timing, and waiting slightly too long before an assaycan permit rapid cytoskeletal organization and strengthening responses that anchor cellsso tightly to the substrate that they cannot be detached without being destroyed. Never-theless, centrifugal detachment assays arguably provide the most rigorous quantitativedata.

Methods for quantification of cell-to-cell adhesion generally depend on determining therates of reaggregation of dissociated cells (see UNIT 9.3). Although it is also possible toestimate strengths of adhesion by determining the force needed to separate two cells usingmicroneedles, aggregation assays are the norm. These assays are conceptually related tothe platelet aggregometer assays used routinely in clinical laboratories to measure plateletadhesion (aggregation). An added feature with nucleated cells, however, is that multipletypes of adhesion molecules can often be involved in any particular cell-cell adhesiveevent. Three commonly observed types of adhesion molecule are the calcium-dependentcadherins, calcium-independent molecules such as N-CAM and other immunoglobulin-repeat molecules, and integrins, though other adhesion molecules may also be involved.

CHARACTERIZATION OF CELL ADHESION MECHANISMS

Once the adhesive characteristics of a cell toward a particular cellular or substrate adhesivesystem are established, a variety of inhibitors or modulators of adhesion can be tested inorder to better define the adhesion system. Examples include function-blocking antibod-ies against the many dozens of known adhesion molecules to determine which are requiredfor an adhesive event, competitive peptide inhibitors that target the active sites of adhesionreceptors, and pharmacological and ionic activators and inhibitors. The latter categoriesinclude phorbol esters or Mn2+ to activate certain integrins, and chelators for depletingCa2+ to inhibit cadherins and integrins. For routine analyses of integrins, the pertinentreceptor(s) involved in a particular adhesive function can be identified by targeting a broadclass of integrins first—for instance using an antibody that inhibits all β1 or all αv

integrins—and then narrowing down the possible candidate receptors involved in aparticular function by using more specific antibodies within that class (e.g., anti-α5 withinthe β1 integrin class). Monoclonal antibodies against integrins are widely available. Forcadherins, specific antibodies against classical but not novel cadherins are also available,as are various cell biological approaches as described in this chapter. Reagents foranalyzing other types of adhesion molecules should become increasingly available fromcommercial sources.

PROCEDURES DESCRIBED IN THIS CHAPTER

Two semiquantitative assays for measuring cell adhesion to a substrate are provided inUNIT 9.1. The first is a cell spreading assay in which adhesion is evaluated by determining


9.0.7

Cell Adhesion

the percentage of cells that spread on a substrate, using microscopy to count spread cells.The second assay quantitates cell attachment after washing out nonattached cells, usinga colorimetric protocol for quantification. The relative advantages of each of these widelyused assays are compared.

The McClay centrifugation assay for directly quantitating the strength of adhesion of cellsto a substrate is described in UNIT 9.2. This assay determines the proportion of cells detachedby the graded application of centrifugal force. A new variation of the original methodprovides a simple method for quantitation by counting numbers of stained cells under alight microscope. This simplification of the assay should further enhance the applicabilityand popularity of this assay. UNIT 9.2 also provides a helpful in-depth conceptual discussionof the issues involved in quantitating cell adhesiveness.

In UNIT 9.3, the original discoverer of the important cadherin cell-cell adhesion systemprovides an overview of cadherins, as well as detailed laboratory protocols for assayingcell-cell adhesion. Both quantitative and qualitative methods for identifying cadherinfunction are presented. Besides providing complete protocols for assaying cell-celladhesion of a single cell type in short- and long-term cultures, this unit describes a methodfor assaying the sorting-out behavior of different types of cells in a mixed-cell aggregationculture. In addition, a variety of approaches for the detection and characterization ofcadherin and associated catenin systems are discussed, along with dominant-negativeinhibitor approaches to characterizing this centrally important cell-cell adhesion system.

UNIT 9.4 describes current experimental approaches used to analyze integrin functions inmediating adhesive and other interactions of cells with specific substrates. This unit alsoprovides detailed procedures for in vitro analyses of binding interactions between purifiedintegrins and their ligands.

UNIT 9.5 provides a variety of protocols for studying the major group of calcium-inde-pendent cell-cell adhesion proteins termed the immunoglobulin superfamily of celladhesion molecules (IgSF-CAMs). This superfamily contains over 100 adhesion mole-cules. After describing how to purify IgSF-CAMs from tissues or culture supernatants,this unit describes a number of approaches to characterizing their biological functions invitro and in living cells. Assays with fluorescent beads provide ways to mimic and toanalyze IgSF-CAM adhesive functions in isolation or in interactions with cells. Comple-mentary approaches using various transfection, adhesive-substrate, inter-molecular inter-action, and functional disruption analyses provide a powerful collection or tools tounderstand the roles and mechanisms of this major class of adhesion proteins.

UNIT 9.6 turns to dynamic analyses of cell adhesion. Cell adhesion often consists of sequentialsteps in dynamic processes that can be difficult to visualize and experimentally analyze interms of mechanisms using simple cell attachment protocols. For example, leukocytes in theblood stream do not simply settle by gravity onto surfaces such as endothelial cells or exposedmatrix in vessel walls, but they instead interact dynamically in the presence of flow and shearstress. In addition, some adhesive events that occur rapidly are difficult to study in static assaysbut can be analyzed effectively using dynamic flow methods. UNIT 9.6 describes methods forsuch analyses using commercially available flow chambers in which cells being carried insuspension attach in the presence of laminar flow. Flow assays can measure cell adhesioneither to a monolayer of endothelial cells or to purified extracellular matrix proteins attachedto a substrate. The resulting data obtained in the form of video microscopy recordings canthen be analyzed using approaches described in the second basic protocol.

Further supplements will provide protocols for analyzing other types of cell adhesion systems.


9.0.8

Introduction

LITERATURE CITED

Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., and Watson, J.D. 1994. Cell junctions, cell adhesions,and the extracellular matrix. In Molecular Biology of the Cell, 3rd ed, pp. 949-1009. Garland Publishing,New York.

Burridge, K., Chrzanowska-Wodnicka, M., and Zhong, C. 1997. Focal adhesion assembly. Trends Cell Biol.7:342-347.

Chothia, C. and Jones, E.Y. 1997. The molecular structure of cell adhesion molecules. Ann. Rev. Biochem.66:823-862.

Clark, E.A. and J.S. Brugge. 1995. Integrins and signal transduction pathways: The road taken. Science268:233-239.

Edelman, G.M. and J.P. Thiery (eds.). 1985. The Cell in Contact: Adhesions and Junctions as MorphogeneticDeterminants. John Wiley & Sons, New York.

Edelman, G.M. and Crossin, K.L. 1991. Cell adhesion molecules: Implications for a molecular histology.Ann. Rev. Biochem. 60:155-190.

Gumbiner, B.M. 1996. Cell adhesion: The molecular basis of tissue architecture and morphogenesis. Cell84:345-357.

Hay, E.D. (ed.). 1991. Cell Biology of Extracellular Matrix. Plenum, New York.

Jockusch, B.M., Bubeck, P., Giehl, K., Kroemker, M., Moschner, J., Rothkegel, M., Rudiger, M., Schluter,K., Stanke, G., and Winkler, J. 1995. The molecular architecture of focal adhesions. Ann. Rev. Cell Dev.Biol. 11:379-416.

Kirkpatrick, C. and Peifer, M. 1995. Not just glue: Cell-cell junctions as cellular signaling centers. Curr. Opin.Genet. Devel. 5:56-65.

Lafrenie, R.M. and Yamada, K.M. 1996. Integrin-dependent signal transduction. J. Cell Biochem. 61:543-553.

Richardson, P.D. and Steiner, M. 1995. Principles of Cell Adhesion. CRC Press, Boca Raton, Fla.

Schwartz, M.A., Schaller, M.D., and Ginsberg, M.H. 1995. Integrins—Emerging paradigms of signaltransduction. Ann. Rev. Cell Dev. Biol. 11:549-599.

Takeda, H., Nagafuchi, A., Yonemura, S., Tsukita, S., Behrens, J., and Birchmeier, W. 1995. V-src kinaseshifts the cadherin-based cell adhesion from the strong to the weak state and β-catenin is not required forthe shift. J. Cell Biol. 31:1839-1847.

Takeichi, M. 1990. Cadherins: A molecular family important in selective cell-cell adhesion. Ann. Rev.Biochem. 59:237-252.

Tamura, M., Gu, J., Matsumoto, K., Aota, S., Parsons, R., and Yamada, K.M. 1998. Inhibition of cell migration,spreading, and focal adhesions by tumor suppressor PTEN. Science 280:1614-1617.

Wolfsberg, T.G. and White, J.M. 1996. ADAMs in fertilization and development. Dev. Biol. 180:389-401.

Woods, A. and Couchman, J.R. 1998. Syndecans: Synergistic activators of cell adhesion. Trends. Cell Biol.8:189-192.

Yamada, K.M. and Geiger, B. 1997. Molecular interactions in cell adhesion complexes. Curr. Opin. Cell Biol.9:76-85.

Yamada, K.M. and Miyamoto, S. 1995. Integrin transmembrane signaling and cytoskeletal control. Curr.Opin. Cell Biol. 7:681-689.

Yap, A.S., Brieher, W.M., and Gumbiner, B.M. 1997. Molecular and functional analysis of cadherin-basedadherens junctions. Ann. Rev. Cell Dev. Biol. 13:119-146.

Kenneth M. Yamada


9.0.9

Cell Adhesion

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UNIT 9.2Quantitative Measurement of Cell AdhesionUsing Centrifugal ForceThe following protocol was developed in order to study the biophysical sequence of eventsin cell-substrate adhesion, and it can also be adapted to study cell-cell adhesion. Themethod allows quantification of the weak association between cells and their substratumat 4°C, thereby giving a measure of the receptor-ligand affinity only. The approach alsoallows measurement of the ATP-dependent events that strengthen adhesion and thatinvolve the cytoskeleton following initial binding. This is done by incubating cells incontact with substrate at 37°C for increasing periods of time. Centrifugal force is the onlyshear force involved in the assay; thus the strength of any adhesion that resists this appliedforce can be accurately measured. The original centrifugal adhesion assay developed byMcClay et al. (1981) used radioactively labeled cells in a rather cumbersome procedureto quantify adhesion. The protocol given here is much easier than any previously publishedversion. At the same time, the assay remains highly quantitative and has simple innova-tions that can accommodate many kinds of adhesion studies.

The procedure is as follows: 96-well polyvinyl chloride (PVC) plates with flat bottoms arecoated with the substrate target for the cells. The wells are then treated with BSA or anothernonadherent protein that blocks nonspecific binding sites. Cells are added to the PVC plateson ice, the wells are filled to the brim, and then sealed with clear packing tape. The idea isto enclose each well as a sealed, fluid-filled compartment without any air bubbles. Thecells are then gently centrifuged into contact with the substrate. To measure cell-substrateaffinity alone, the wells are kept at 4°C, flipped upside down, and centrifuged to providea defined dislodgement force for the cells (Fig. 9.2.1). Using replicates challenged withdifferent centrifugal forces one can determine the relative avidity of cells for a substrate.If cells are incubated at 37°C after being centrifuged onto the substrate, one can follow theprogress of adhesive strengthening due to cytoskeletal engagement. This process generallyoccurs rapidly, and progress can be monitored by this assay.

There are many ways to quantify cells adhering to the substrate. Currently, the simplestis to image the well bottom and count cells bound relative to controls and to the total

Contributed by David R. McClay and Philip L. HertzlerCurrent Protocols in Cell Biology (1998) 9.2.1-9.2.10Copyright © 1998 by John Wiley & Sons, Inc.

applysubstrate

applycells

seal andcentrifugeto contact;invert plate

centrifugeto apply

dislodgement force

count cellsbound

1 to 35 × g1 to 500 × g

Figure 9.2.1 Centrifugal force adhesion assay with light microscopic readout.

9.2.1

Cell Adhesion

number of cells originally added. Other approaches can easily be adapted; however, thismethod provides a simple visual measure of adhesion that is quite easy to learn, is easilyadapted by any laboratory at low cost, and allows for a number of variations to be applied,including morphometrics (see Background Information).

BASICPROTOCOL

CENTRIFUGATION CELL ADHESION ASSAY

Materials

96-well flat-bottom polyvinyl chloride (PVC) plates (Falcon)Substrate molecule of interest in PBS40 mg/ml BSA (fraction V; Sigma) in PBSPBS (APPENDIX 2A)Tissue culture medium without FBSDissociated cells of interest, suspended at 5–10 × 105/ml in calcium-free

physiological solution (see Critical Parameters, discussion of suitable media)

Clear packing tape (3M Scotch 375, 4.8 cm wide, although any clear packing tapewill probably work)

Microtiter plate support templateLow-speed refrigerated cell centrifuge with microtiter plate carrier

1. Cut away the sides of a 96-well flat-bottom PVC plate with scissors, and then cut theplate into a 3 × 8–well rectangle.

Only the six wells in the middle are used. Since the assay involves filling the wells over thebrim to eliminate air bubbles, some of the medium will spill into the surrounding emptywells. Plates can be cut into other configurations depending upon the centrifuge carrier,the microscope, or other variables that are particular to the lab.

2. Add 50 µl substrate per well, leaving blank wells for 100% attachment (no substrateor blocking) and for background binding (no substrate). Use at least three replicatesfor each parameter tested. Incubate ∼30 min at room temperature.

Polyvinyl chloride has a natural avidity for many proteins.

As a control, determine how much of the substrate protein is attached to the plate. In thefirst set of experiments with a substrate protein or peptide, determine the thresholdconcentration for binding (relative to the BSA control wells and at a low g force), and thenoperate just above that threshold in subsequent experiments (see Critical Parameters).

3. Wash three times with 100 µl PBS. Then block substrate-coated and backgroundbinding wells (but not 100% attachment control wells) ∼30 min with 50 µl of 40mg/ml BSA at room temperature. Wash three times with 100 µl PBS, flicking thecontents of the wells into the sink to eliminate the fluid after each wash.

It is important that the background control well does not bind cells. BSA treatment shouldblock all spaces not covered by the substrate molecule. Typically 100% of cells bind to theuntreated wells, and <1% bind to the background wells. Although this varies according tothe cell type used and the dislodgement force applied, in most cases the conditions can beadjusted so that <1% of cells bind to the BSA-blocked wells. This leaves the test substratewith a wide range in which to examine adhesion avidity.

If desired, poly-L-lysine can be added to the 100% attachment control well (no substrate,no blocking) to bind cells even more tightly.

4. Place the plate on ice. Add 100 µl tissue culture medium to all wells, add 100 µl cellsuspension (5–10 × 104 cells), and then fill all wells with another 100 µl tissue culturemedium.

Fewer cells can be added, especially if fluorescent cells are used for later quantification.For fluorescent cells, good results can be obtained with as few as 1000 cells per well. Also,

Current Protocols in Cell Biology

9.2.2

QuantitativeMeasurement of

Cell AdhesionUsing Centrifugal

Force

it is possible to employ two different fluorescent colors and unstained cells in the samewell, so that two experimental cell types can be compared to a control cell population underexactly the same conditions.

Medium and cells should be added on ice to prevent cells from attaching and adheringprematurely. There should be a positive meniscus bulging above the top of the well, asexcess fluid is necessary to seal the well without any air bubbles.

5. Gently lay a piece of clear packing tape over the wells, and place the plate on amicrotiter plate support. Apply the tape, starting from one end and squeegeeing theexcess medium to spill over into the empty wells next to the test wells. Press downon the tape to make sure it adheres to the PVC surrounding each well.

The microtiter plate support is a rubber or metal template that holds the PVC plate whileit is being sealed. A firm backing is necessary to support the pressure applied when sealingthe tape. A metal plate can be made by a machine shop. Its thermal conductance keeps thewells cold during the manipulation and sealing procedures. If metal supports are unavail-able, a 96-well plate can be used as a mold to make a plastic support device.

With a little practice, this maneuver can be done without introducing any bubbles into thesealed wells. If sealing is done correctly, each well should now be a completely filled,enclosed chamber, and should survive centrifugal forces up to ∼500 × g without leaking.Some cells will be lost in the process of sealing, but that will not add an error factor to theanalysis because of the way cells are counted against controls in the assay.

6. Centrifuge cells into contact with the substrate for ∼3 min at 35 × g, 4°C, in alow-speed refrigerated centrifuge with a microtiter plate carrier.

Some larger cells can be allowed to settle onto the substrate at 1 × g. If cells are centrifugedonto the substrate, ∼3 min of centrifugation will place 100% of the cells into contact withthe substrate.

If this is done at 4°C, receptor-ligand combinations are brought into contact. As long asthe plate is left at 4°C, this receptor-ligand affinity can be measured independent ofcytoskeletal contributions.

One can substitute a big centrifuge bucket as a swinging bucket carrier if a microtiter platecarrier is not available.

7. Optional: To measure strengthening events in adhesion, simply move the plate, withcells in contact with the substrate, to a 37°C water bath and float the plate for thedesired period of time.

For many cell types, incubations <5 min are sufficient to convert the weak receptor-ligandbinding into a highly strengthened adhesion that is strongly resistant to centrifugal shearforces.

8. Invert the plate and centrifuge the cells in the inverted position using a microtiterplate carrier, 5 min at the desired speed. Return the plate to ice, keeping it in theinverted position.

This should dislodge cells if enough centrifugation force is applied and if the cells are notstrongly adhering. Obviously there are a number of parameters that can be controlled inthis step: e.g., duration of contact at 37°C, rate of dislodgement, force of centrifugation,and rate of strengthening.

9. Place the wells on the stage of a compound microscope, and count cells using a 10×objective. Subtract background binding from substrate binding, and determine thepercent binding compared to the 100% attachment control.

Imaging software can be used to capture frames and automatically count the cells in eachframe. To increase accuracy, set up the microscope so it will image a well prior tocentrifugation, then return to the same field after centrifugation to assess adhesion.


9.2.3

Cell Adhesion

COMMENTARY

Background InformationEarly cell adhesion assays used cell aggre-

gation as a semiquantitative measure. Theseassays were useful for studying some adhesivephenomena, but suffered from an inability toquantify the sequence of adhesion. Attempts toquantify adhesion have used viscometers, cellparticle counters, spectrophotometers, radioac-tivity, and other approaches. In most cases,adhesion assays were limited in that they re-quired cells to associate rather tightly with oneanother in order to survive unknown, or poorlydefined, shear forces intrinsic to the assay.

This centrifuge assay was designed to use asingle shear force of defined magnitude. Theforce operating on a cell can be easily calcu-lated, providing an impression of the strengthof an adhesion. This approach, though quanti-tative, is still not perfect. For example, onewould really like to measure the “on rate” as anadhesion is formed, but this is an exceedinglydifficult parameter to measure. Also, the assaydescribed here measures the proportion of ad-herent cells within a population, rather than thebehavior of individual cells. Single cell assaysmay offer advantages not offered by the assaygiven here.

The present assay is based on previouslypublished versions (McClay et al., 1981; Lotzet al., 1989; Burdsal et al., 1991, 1994) in whichcentrifugal force is used as a dislodgementforce (FD) for the cells. This assay is highlymodified and simplified compared to the origi-nal assays, but enjoys a better and more versa-tile capacity for quantification.

The following relationship is important forunderstanding where force fits into the adhe-sion process. As measured in the centrifugaladhesion assay, FD = (ρcell − ρmedium) × Vcell ×RCF, where FD is the dislodgement force(dynes/cell) tending to pull the cell from thesubstrate, (ρcell − ρmedium) is the specific densitydifference between the cell and the medium(usually 1.07 g/cm3), Vcell is the volume of thecell, and RCF is the relative centrifugal force(McClay et al., 1981). Because of the smalldifference in specific density between the fluidand the cell, cells in culture are little affectedby alterations at 1 × g, since in reality these cellsare already experiencing microgravity.

The assay as presented above allows for veryprecise comparisons between cells that are onlyslightly different from one another. For exam-ple, if one cell population is transfected with aconstruct and the other population is a non-

transfected control, the two can be tested in thesame well (with one of the populations fluores-cently tagged) to accurately compare adhesiveperformance.

Most other cell-substrate assays have a cer-tain unknown shear force that can confoundquantification, especially when one wants sim-ply to measure receptor-ligand interactions.The simple “stick-and-wash” assay, in whichone allows cells to bind to the substrate for aperiod of time and then washes the wells, maybe simpler to use than the assay presentedabove, but suffers from several problems. It hasan undefined shear force (washing) that is dif-ficult to control accurately. Stick-and-wash as-says also cannot measure the initial receptor-li-gand interactions, because cells must adhere atleast somewhat tightly to the substrate in orderto survive washing. Thus, the adhesion meas-ured in a stick-and-wash assay is actually bothadhesion and engagement of the cytoskeleton.Often such assays are completed hours aftercells were added to wells, so that any numberof postadhesion events could occur beforemeasuring “adhesion.” In contrast, the presentassay allows measurement of the progressionof several adhesion events separately, andtherefore has the capacity to examine severalparameters in the sequence of forming an ad-hesion. It should be mentioned that for morethan two decades the simpler “stick-and-wash”assay was successfully employed by a numberof laboratories to find and characterize most ofthe known adhesion molecules. The presentassay is useful for detailed structure-functionanalyses of those molecules.

An additional parameter that can be meas-ured with the centrifugal adhesion assay is therelationship between adhesiveness and cellphenotype. Since the cells are counted by mi-croscopy, one can also score cell phenotypicproperties (e.g., spreading or motility). Thisadds to the versatility of the assay. The assaycan be used, with minimal adaptation, for awide variety of cell types, including cells fromseveral phyla, tumor cells, and cells of all stagesof embryonic development.

Initial bindingIn practice there are many events in the

establishment of an initial adhesion. There isan on rate by which the first receptor-ligandinteractions occur. Then there is a rate of re-cruitment of additional cellular adhesion mole-cules to the site of adhesion. This parameter has


9.2.4



Force

been measured, and is related to the rate ofdiffusion of the protein in the phospholipidbilayer. As long as no additional molecules areadded to the bilayer, the total recruitment ofreceptor-ligand combinations is limited to thetotal population in the membrane at the time ofthe assay. Finally, since the interaction is notcovalent, there is an off rate. Most cell typesbind to substrates with a force that resists 10−6

to 10−5 dynes/cell of applied centrifugal dis-lodgement force. In reality, cells come off thesubstrate when the off rate (increased by cen-trifugal force) exceeds the on rate. Red bloodcells fail to adhere to most substrates at around10−8 dynes/cell. Some cells, such as macro-phages, naturally adhere with a receptor-ligandavidity that is slightly higher than the 10−5

dynes/cell level. When measuring initial bind-ing one must measure the adhesion at very lowsubstrate concentrations. Once the well be-comes supersaturated with multiple layers ofsubstrate, the initial binding loses specificityfor the receptor-ligand combination under in-vestigation. This presumably results from non-specific effects of charge and substrate hetero-geneity.

Cytoskeletal contribution to strengtheningAfter obtaining a measurement of initial

binding (receptor-ligand affinity), a measure-ment can be made of the strengthening of thatadhesive process. This cellular process is re-markably fast. Using this assay, most strength-ening is completed within 5 min at 37°C. Thedegree of strengthening is usually more than 2orders of magnitude, and may be as much as 5to 6 orders of magnitude.

The limitations of this assay come into playduring the strengthening process. Although werecommend a range of 1 to 500 × g for the assay,the centrifuge plate carriers have a maximumsafe speed that cannot be exceeded. Using largebuckets as plate carriers can increase the cen-trifugation speed to ∼4000 rpm. This means thatthe assay can be extended to forces between 1and ∼2500 × g in most centrifuges. In measure-ments using the ultracentrifuge to achieve veryhigh forces, it was found that adherent fi-broblast cells are not released by 15,000 × g,and only 30% are released at ∼59,000 × g (Rich,1978). Thus, for practical measurements ofstrengthening, one can quantify the early eventsto monitor the rate of strengthening, and onecan study cells with modified cytoskeletons toexperimentally address this mechanism.

Again it should be noted that the “stick-and-wash” assays that are employed in many labo-

ratories simply measure strengthened adhe-sion. In contrast, this is an accurate assay aslong as the rate-limiting step in the adhesionsequence is the adhesive step of interest. Thestick-and-wash assays can be misleading insuch tests. For example, consider a laboratorywith an interest in cytoskeleton contributions.Hopefully, if the stick-and-wash assay is usedwith cytoskeletally impaired cells, the data willindicate that no adhesion has occurred. Thismust assume that the initial binding occurs asnormal, but that the cells then fail to strengthendue to the missing critical cytoskeletal compo-nent. However, if the initial cell-substrate bind-ing step fails to occur properly, the investigatormight be misled into thinking the cytoskeletonis at fault. With the centrifugal assay, it ispossible both to separate initial adhesion fromstrengthening, and to determine the extent ofstrengthening. This application has been veryuseful in a variety of circumstances involvingembryonic cells of different stages, or trans-fected cells with different adhesion deficien-cies.

Biophysical analogy of initial bindingversus strengthened adhesion

There are many biophysical models of ad-hesion that attempt to explain how the cy-toskeleton works to accomplish an increase inadhesive strength by orders of magnitude.Based largely on the knowledge that cells aremalleable, and knowing that cells adhere bynoncovalent receptor-ligand interactions,Dembo and Bell (1987) showed that initialbinding was relatively weak and was similar formost adhesion molecules. Strengthened adhe-sion, on the other hand is highly resistant toshear forces. In the Dembo and Bell model,cells adhering by receptor-ligand interactionsalone simply peel away from the substratebreaking bond by bond. When adhesion isstrengthened by recruitment of the cytoskele-ton into the adhesion complex, there is a rapidand very large increase in adhesive strength. Asa simple analogy of the initial binding versusstrengthened adhesion, consider a piece of dou-ble-stick Scotch tape. The following test is asimple means of demonstrating this principle.

Stick a piece of double-stick Scotch tapeonto a desk. The Mylar backing of the tape ismalleable so that the force needed to initiallystart peeling one end of the tape away from thesubstrate will be fairly small, and will remainfairly constant until the entire strip of tape ispeeled away from the substrate. This is becausethe peeling force is the force necessary to break


9.2.5

Cell Adhesion

the adhesive bonds only at the immediate pointof contact as the tape is progressively peeledaway (Fig. 9.2.2A). The rate of peeling dependsupon the applied force, the number of bonds,the strength of an individual bond, and the onand off rates of the bonds. As the peeling forceis increased, a point is reached when the off rateexceeds the on rate causing the adhesion to failat that peeled interface. In the centrifugal assay,the centrifugal force just below the thresholdpoint of cell removal is a measure of the initialbinding affinity of the cell.

To demonstrate how adhesive strengtheningappears to work, place a microscope slide ontop of the same piece of double-stick tape onthe desk. The tape is the same, the adherenceto the substrate is the same, but now instead ofa malleable backing the glass provides a verystiff backing for the tape. An exposed end ofthe tape peels away rather easily until the edgeof the slide is encountered. Then a force that isorders of magnitude larger must be used to

remove the tape from the substrate. The massiveadditional force needed to lift the tape from thesubstrate is a consequence of the hard backingprovided by the glass slide. Because of thatrigid backing, the entire piece of tape must beremoved all at once. Thus, the entire adhesivesurface of the tape under the glass slide simul-taneously resists the removal force (Fig.9.2.2B).

By analogy, this rigidity is a biophysicalproperty provided by the cytoskeleton that en-ables the adhesion to be strengthened. By co-operatively linking multiple adhesive mole-cules into more of a unit, a vast increase inapparent adhesive strength can be accom-plished without an increase in number of adhe-sion molecules. Mechanically, the cytoskeletoncauses a local stiffening of the adhesion com-plex so that many more adhesion molecules areacting together to cooperatively strengthen theadhesion.

A

B

peeling force

area resisting peeling force

area resisting peeling force

peeling force

Figure 9.2.2 Tape analogy of two states of cell adhesion. (A) Double-stick tape applied to a surfaceis analogous to the adhesive behavior of cells that have only receptor-ligand association with thesubstrate. The adhesive area that resists the peeling force is small, limited to the area between thetwo arrows. (B) Adding a glass slide to the upper surface of the tape creates a situation analogousto the adhesive behavior of cells with cytoskeleton-supported cooperativity of cell-substrateassociation. The stiffened area of tape (membrane) increases the adhesive area that resists thepeeling force.


9.2.6



Force

Obviously, the cortex of the cell is not asrigid as the glass backing. Nevertheless, anycoupling of the cytoskeleton to the group ofadhesion molecules that allows these mole-cules to act as a unit rather than as freelydiffusing molecules, provides an important co-operativity for strengthening. Conveniently,this allows the cell to control its own adhesive-ness by controlling intracellular microcompart-ments (areas where adhesive molecules cancouple with cytoskeleton and other areas wherethe cytoskeletal connections are released). Anability to measure many components of thesedynamic associations is necessary to under-stand how adhesion molecules and cytoskele-ton components work together to provide cellswith a range of adhesive properties. The cen-trifugal adhesion assay can help with suchanalyses.

Critical Parameters

Concentration of substrateAs a rule, the substrate should be applied as

a molecular monolayer on the plate. In the firstset of experiments with a substrate protein orpeptide, determine the threshold concentrationfor binding, and then operate just above thatthreshold in subsequent experiments. If onecalculates the total surface area occupied by onesubstrate molecule, and calculates the total sur-face area of the well bottom, it is a simplecalculation to figure out how much substratewould form a molecular monolayer. For exam-ple, ∼1 µg of fibronectin per well is about theright amount. Some overlapping is assumed,since the protein is filamentous, but the pointis that it takes very little protein to completelycoat the plate. At a concentration of 10 µg/well,the assay loses specificity, presumably becausemany nonspecific factors can now contribute tothe adhesion. Also, if the protein is layered onthe plate, the effects of fibronectin peeling awayfrom fibronectin may confound the assay.

Timing of adhesive strengtheningSignificant strengthening is seen after even

1 min of incubation at 37°C. By 5 min at 37°C,the strengthening of most cells exceeds theability of the cell centrifuge to provide enoughforce to dislodge them, because most bucketcarriers or 96-well plates have a maximum RCFat which they can be centrifuged (usually onthe order of 2500 × g). Note that this level ofstrengthening occurs well before any signifi-cant change can be seen in the cells, and beforethey establish visible focal contacts.

Increments in centrifugal forceFor initial binding, cells can be centrifuged

onto the substrate at ∼35 × g. For dislodgementof cells kept at 4°C, the force can vary from 1to ∼2500 × g. Most cells will maintain adhesionto between 50 and 200 × g, but will be removedby higher g forces. For strengthened adhesions,a good guideline is to use a dislodgement forcethat is twice that necessary to remove the in-itially bound cells, and then ask how long ittakes for the cells to strengthen at 37°C enoughto remain adherent at that force. Usually thattime is <2 min.

Suitable mediaThe selection of medium depends on the cell

type used, and several kinds of media are usedfor each assay. Cells should be dissociatedusing established protocols and media for eachcell type. The cells are then maintained in acalcium-free buffer for counting and handlingduring the short time prior to adding them tothe wells (see discussion of cations below).Finally, tissue culture medium without any fetalbovine serum (or equivalent) should be used asthe cell adhesion test medium. If necessary,fetal bovine serum can be included, providedthat proper controls are done for the contribu-tion of cell adhesive substrates in the serum.

Phosphate buffered saline (PBS) containing40 mg/ml BSA is used as a blocking solutionfor all wells pretreated with the substrate inquestion, as well as for background bindingwells. In the authors’ experience, the BSAeliminates almost all background binding to thewell bottom. PBS without BSA serves as thewashing reagent and as the buffer into whichthe substrates are diluted when initially coatingthe wells.

Role of cationsMany cell-cell interactions require calcium

for some aspect of association. The actual func-tion of calcium in these interactions has a richliterature. For the present case, keeping cells incalcium-free solutions is a useful way to controlthe experiment and to prevent adhesion fromoccuring prematurely. Thus, cells are added incalcium-free saline to the medium in the wells.There is enough calcium in most tissue culturemedia to allow this dilution of calcium in thewell. Note that the calcium-free medium doesnot contain EGTA or any other calcium chela-tor. If the dissociation protocol uses calciumchelators, resuspend the cells in calcium-freemedium without the EGTA after they are dis-sociated.


9.2.7

Cell Adhesion

Recovery period after dissociationIf cells are enzymatically dissociated, they

will need a recovery period to regenerate sur-face adhesion molecules prior to using the cen-trifugation assay. It is preferable to use nonen-zymatic methods for dissociation so that cellsare ready to use without recovery. If a recoveryperiod is necessary, cells can be resuspended incomplete culture medium with serum, and in-cubated in flasks at a rotation speed >70 rpm.This provides a chronic fluid shear force thatprevents cells from adhering, yet allows themto recover from enzymatic treatments. Thelength of recovery varies according to cell type,but 4 hr at 37°C is a good first approximation.The cells should then be washed into calcium-free, serum-free medium for the assay.

Fluorescent molecules for labelingMany fluorescent tags are available, though

some work better than others. Rhodamineisothiocyanate (RITC) is the authors’ firstchoice simply because it works on all cells andis slow to fade. The method of labeling issimple. Take ∼1 mg of RITC (a bit on the endof a spatula) and add it to 50 µl dimethylsul-foxide (DMSO). Add the RITC/DMSO to cal-cium-free medium (without phenol red as a pHindicator) until the color of the medium barelyturns pink. If the medium turns red, too muchRITC has been added and the medium is toxic.Allow the cells to accumulate the RITC fromthe slightly pink medium, and they will behealthy. Usually 30 min or less is all that isnecessary for labeling.

Several markers can be used as a secondlabel. Fluorescein isothiocyante (FITC) tendsto be a bit difficult, as batches of FITC loseactivity quickly. The fluorescein quenches eas-ily as well. The easiest to use is Hoechst dye(33342 or 33258), which is easily loaded intocells, stains nuclei brightly, and is retained well.To label the cells, dissolve a small amount ofHoechst (or DAPI) dye in aqueous medium,add it to the cells at ∼1:200, and incubate 30min. Other dyes such as calcein AM or acridineorange can also be used to label other popula-tions of cells. These dyes are easily loaded, andhave good retention (i.e., do not bleed intounlabeled cells). To label cells, add 10 µl of afreshly prepared dye solution (up to 1 mg in 50µl DMSO) to 100 ml cells in aqueous medium.Incubate 30 min, wash, and check labeling.Adjust as necessary, as labeling varies with celltype. High dye concentrations should beavoided because they may be toxic, and because

most dyes are cumulative (i.e., they will gradu-ally be accumulated from a weak solution).

TroubleshootingThere are three potential pitfalls to be

avoided in the assay. All three can be managedwith relative ease, but some tips are offeredbelow if these problems confound the assay.

Bubbles from improper sealingThe biggest problem encountered by the

novice is the introduction of bubbles of air intothe chamber when the wells are sealed. Thebubble will act like a bulldozer and sweep cellsoff the substrate when the wells are inverted.To avoid this, it may take some practice sealingthe wells. The easiest way to put the tape on isto place the wells in a rubber support, which actsas a backstop when the tape is pressed down. Thetape is then rolled onto the wells, and anyremaining medium is squeegeed by rubbing thetop either with fingers or a flat surface.

Bubbles generated during the warmingincubation

If the sealed plate is incubated at 37°C for>5 min, air bubbles are often introduced. Toavoid this, the plates can be sealed after the37°C incubation. For long incubations, fill thewells with 100 µl of medium plus 100 µl of cellsuspension (2/3 full). After the incubation,place the plates on ice and add 100 µl of me-dium to brim the wells. Add the tape and do thedislodgement step. In practice, this is really aneasy step; however, it does introduce a potentialerror in that the late addition of medium adds ashear force other than centrifugal force to thesystem. One can easily control for this by ex-amining the wells both prior to and after top-ping them off.

Quantifying cellsThe cells will not be uniformly distributed

on the bottom of the wells, which can poten-tially cause a sampling problem. This can becompletely avoided by comparing an experi-mental population of cells, labeled with onedye, against a reference control population ofcells, labeled with a different dye, in the samewell. It is then possible to detect very smalldifferences that might exist between the twocell populations. To count cells, it is simplestto capture images of the well bottoms, andeither count the cells manually or use a com-puter-generated macro to automatically countthe cells in the field.


9.2.8



Force

Anticipated ResultsWith most cell types, two basic types of

results will be obtained. Measuring adhesion asa function of g force will generate the twocurves seen in Figure 9.2.3A. The cells adher-ing by a receptor-ligand interaction alone (4°C)will survive the dislodgement force until a cer-tain force is reached and then there will be arather sharp drop in their ability to resist thedislodgement force. On the other hand, if thecells are subjected to a brief incubation at 37°Cwith time held constant, they will tend to resistremoval. Figure 9.2.3B shows an example ofexpected results when measuring adhesion asa function of time at 37°C, with the dislodge-ment force held constant.

Time ConsiderationsA typical assay requires 1 hr to complete.

Extra time is required if the cells must recoverafer dissociation or if strengthened adhesion ismeasured.

Literature CitedBurdsal, C., Alliegro, M.C., and McClay, D.R. 1991.

Echinonectin as a substrate for adhesion duringdevelopment of the sea urchin embryo. Dev. Biol.144:327-334.

Burdsal, C.A., Lotz, M.M., Miller, J., and McClay,D.R. 1994. A quantitative switch in integrin ex-pression accompanies differentiation of F9 cellstreated with retinoic acid. Dev. Dynamics201:344-353.

A

B

% c

ells

adh

erin

g

100

0

50

g

Time at 37°C (min)

preincubation at 4°C

preincubation at 37°C

1 2 3 4 5 6 7

% c

ells

adh

erin

g

100

0

50

1000

Figure 9.2.3 Theoretical data. (A) Force (in g) required to dislodge cells preincubated at 4°C and37°C. (B) Time required to stabilize initial adhesion.


9.2.9

Cell Adhesion

Dembo, M. and Bell, G.I. 1987. The thermodynam-ics of cell adhesion. Curr. Top. Membr. Transp.29:71-89.

Lotz, M.M, Burdsal, C.A., Erickson, H.P., andMcClay, D.R. 1989. Cell adhesion to fibronectinand tenascin: Quantitative measurements of in-itial binding and subsequent strengthening re-sponse. J. Cell Biol. 109:1795-1805.

McClay, D.R., Wessel, G.M., and Marchase, R.B.1981. Intercellular recognition: Quantitation ofinitial binding events. Proc. Natl. Acad. Sci.U.S.A. 78:4975-4979.

Rich, A.M. 1978. Substratum amd solution parame-ters of cell adhesion. Ph.D. thesis. University ofNorth Carolina, Chapel Hill.

Contributed by David R. McClay and Philip L. HertzlerDuke UniversityDurham, North Carolina


9.2.10



Force

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UNIT 9.4Analyzing Integrin-Dependent Adhesion

This unit describes methods for the analysis of integrin-ligand binding in both cell-basedassays (see Basic Protocol 1) and solid-phase assays (see Basic Protocol 2). A majorapplication of cell adhesion assays is in investigating whether a certain cell type canadhere to a specific adhesive substrate, and, if so, which receptors are involved. Particu-larly if the substrate is a matrix component (e.g., fibronectin), members of the integrinfamily are likely to play a dominant role in adhesion. Procedures are described here forassessing which integrins are involved in this process. A detailed analysis of ligandrecognition by individual integrins can be performed using a solid-phase receptor-ligandbinding assay. The unit also contains support protocols for integrin purification (seeSupport Protocol 1) and coupling of antibodies to Sepharose for use in this purification(see Support Protocol 2), as well as for biotinylation of integrin ligands to be used in thesolid-phase assay (see Support Protocol 3).

BASICPROTOCOL 1

ANALYZING INTEGRIN-DEPENDENT ADHESION IN CELL-BASEDASSAYS

Cell-based assays for integrin-dependent adhesion are conducted in the same manner asthe spreading and attachment assays described in UNIT 9.1, except that antibodies tointegrins (see Table 9.4.1), peptides, or other reagents are included to identify theintegrin(s) and characterize the molecular events associated with adhesion. Function-blocking monoclonal antibodies are the most useful reagents for these analyses becauseof their high degree of specificity. If only a single integrin is involved in mediatingadhesion, antibodies to either the α or β subunit should abrogate cell adhesion. Forexample, antibodies to either α5 or β1 should completely block HT-1080 fibrosarcomacell attachment and spreading on fibronectin, showing that this interaction is mediatedsolely by α5β1 (Akiyama et al., 1989). If multiple integrins are involved, a combinationof monoclonal antibodies will be required to block adhesion (Mould et al., 1990, 1994).Although peptides and other inhibitors have, in general, less specificity than monoclonalantibodies, they can be useful for determining what amino acid sequence in the ligand isrecognized by the integrin. For example, CS1 peptide inhibits A375-SM melanoma cellattachment to fibronectin, demonstrating that the CS1 sequence is involved in adhesionto this substrate.

Materials

Adhesion molecule of interestDulbecco’s phosphate-buffered saline (DPBS, Life Technologies; also see

APPENDIX 2A)10 mg/ml heat-denatured BSA solution (UNIT 9.1)Cells of interestDMEM/HEPES: Dulbecco’s modified Eagle medium (Life Technologies;

APPENDIX 2B) with 25 mM HEPES, gassed with 5% to 10% CO2

Inhibitor: integrin-specific monoclonal antibody (see Table 9.4.1) or peptide,dissolved in DPBS at appropriate concentration

96-well tissue culture microtiter plate

Additional reagents and equipment for counting cells with a hemacytometer(UNIT 1.1) and spreading or attachment cell-substrate adhesion assays (UNIT 9.1)

1. Prepare the 96-well plate by coating the wells with the adhesion molecule of interestdiluted to the appropriate concentration in DPBS and blocking with 10 mg/mlheat-denatured BSA solution (see UNIT 9.1, Basic Protocol 1, steps 1 to 4).

Supplement 15

Contributed by A. Paul MouldCurrent Protocols in Cell Biology (2002) 9.4.1-9.4.16Copyright © 2002 by John Wiley & Sons, Inc.

9.4.1

Cell Adhesion

Unless the level of adhesion is low, use a concentration of adhesion molecule that promotes50% to 70% maximal adhesion for coating the wells.

2. Prepare a suspension of the cells of interest at a density of 2 × 105 cells/ml in warmDMEM/HEPES gassed with 5% to 10% CO2. Incubate cells in an open tube 10 minat 37°C in a CO2 incubator.

3a. For cell spreading assay: Remove BSA solution from the 96-well plate, wash thewells with 100 µl DPBS, and add 50 µl DPBS containing the inhibitor at twice thedesired final concentration to each well. Add 50 µl cell suspension to each well.

Table 9.4.1 Monoclonal Antibodies Suitable for Use in Cell Adhesion Assays

Integrinsubunita Ligandb mAbc Function-

blocking Supplier

α1 COL/LN 5E8D9TS2/7

YesNo

Upstate BiotechnologySerotec

α2 COL/LN Gi916B4

YesNo

Beckman CoulterSerotec

α3 FN/COL/LN P1B5M-Kid2

YesNo

ChemiconBeckman Coulter

α4 FN/VCAM-1 HP2/144H6

YesNo

SerotecSerotec

α5 FN JBS5VC5mAb 16

YesNoYes

SerotecPharmingenK.M. Yamadad

α6 LN GoH34F10

YesNo

SerotecSerotec

α9 TN Y9A2 Yes Chemicon

β1 4B4K20mAb 13

YesNoYes

Beckman CoulterBeckman CoulterK.M. Yamadad

αL ICAM-1, -2, -3 MHM 24BV17

YesNo

DakoChemicon

αM FG, ICAM-1 ICRF 44LM11

YesNo

SerotecChemicon

β2 MHM 23 Yes Dako

αV VN/FN/FG/TSP 69-6-5P3G8

YesNo

Beckman CoulterChemicon

β3 SZ21PM6/13

YesNo

Beckman CoulterSerotec

β4 ASC-3450-90

YesNo

ChemiconSerotec

β5 P1F6 Yes Life Technologies

aNote that β1 associates with α1-α9, β2 associates with αL and αM, β1, β3, and β5 associate with αV, and β4associates with α6; β2 integrins are expressed only on leukocytes, but most other integrins are widely expressed.bAbbreviations: COL, collagen; FG, fibrinogen; FN, fibronectin; ICAM, intercellular adhesion molecule; LN,laminin; TN, tenascin, TSP, thrombospondin; VCAM, vascular cell adhesion molecule; VN, vitronectin.cAll mAbs, with the exception of GoH3, mAb 16, 69-6-5, and mAb 13, are mouse anti-human. GoH3, mAb 16,69-6-5 and mAb 13 are rat anti-human. See above list of suppliers for antibodies to other species. Antibodiesshould be stored in small aliquots at −70°C.dAvailable by Materials Transfer Agreement from K.M. Yamada, National Institutes of Health.


9.4.2

AnalyzingIntegrin-

DependentAdhesion

3b. For cell attachment assay: Mix an aliquot of the cell suspension with an equal volumeof DPBS containing the inhibitor at twice the final concentration. Incubate 15 to 30min at 37°C. Remove BSA solution from the 96-well plate, wash the wells with 100µl DPBS, and add 100 µl of the cell/inhibitor mixture to each well.

In the authors’ experience, a final mAb concentration of 10 �g/ml, or 1:500 dilution ofascites, gives a maximal inhibitory effect. However, the concentration of mAb to achieve amaximal level of inhibition should be tested using a range of mAb concentrations. Animportant consideration is that the mAbs should be azide-free. Many commercially avail-able mAbs contain sodium azide as a preservative. This is toxic to all cells and will inhibitattachment and spreading. Therefore it is essential that the sodium azide be removed bydialysis into an appropriate buffer such as DPBS.

Peptides can be used in place of mAbs to analyze the contribution of integrins to celladhesion. However, these are generally less useful than mAbs because only for a smallnumber of integrins have specific inhibitor peptides been described. Examples are CS1(DELPQLVTLPHPNLHGPEILDVPST) for �4�1 and �4�7, GACRRETAWACGA for �5�1,and KQGADV for �IIb�3. RGD peptides (e.g., GRGDS) are of broad specificity and inhibit�5�1, �IIb�3, and all �V integrins. However, RGD peptides may be useful as a “firstscreen” to determine if receptors of this class are important for adhesion, or alternativelyto confirm their involvement if cell adhesion is inhibited by, for example, mAbs to �5 or �V.Concentrations of peptides required to inhibit cell adhesion are generally higher than formAbs: typically 0.1 to 1 mg/ml. Higher concentrations are normally required for inhibitingcell attachment than for inhibiting cell spreading. GRGDS, KQGADV, and CS1 peptidesare available from Bachem.

4. Incubate, fix, and analyze for spreading assay (see UNIT 9.1, Basic Protocol 1, step 8to 13) or for attachment assay (see UNIT 9.1, Basic Protocol 2, steps 8 to 18).

It is essential that the effect of each mAb on cell attachment or spreading be compared withappropriate controls. The best controls to use are mAbs that are noninhibitory against theintegrins under test—e.g., K20 for �1 (see Table 9.4.1). However, if these are not available,the following controls can be used: (1) wells to which no mAbs are added (DPBS only);(2) nonimmune mouse or rat IgG or ascites; (3) irrelevant isotype-matched mAb controls.Control peptides (e.g., GRGES) should be used in parallel with the authentic peptides toexclude any toxic or nonspecific effects.

If a partial inhibitory effect is observed with several different anti-integrin mAbs, the assaycan be repeated using two or more mAbs in combination to analyze the relative contributionof each integrin to cell adhesion.

BASICPROTOCOL 2

ANALYZING INTEGRIN-LIGAND INTERACTIONS IN SOLID-PHASEASSAYSThis protocol describes a simple integrin-ligand binding assay in which the integrin isadsorbed to the wells of an ELISA plate. The plate is blocked using BSA (to reducenonspecific binding of the ligand) and then biotin-labeled ligand is added. After washingto remove unbound ligand, bound ligand is detected by addition of an avidin-peroxidaseconjugate followed by a colorimetric detection step. An example of an application of thisprocedure would be use of biotinylated fibronectin or fibronectin fragments as ligandsfor the integrin α5β1.

Materials

Purified integrin (see Support Protocol 1)Dulbecco’s phosphate-buffered saline (DPBS, Life Technologies; also see

APPENDIX 2A)Blocking solution (see recipe)Biotin-labeled ligand (see Support Protocol 3)Binding buffer (see recipe)


9.4.3

Cell Adhesion

ExtrAvidin peroxidase reagent (Sigma)ABTS reagent (see recipe)2% (w/v) SDS in water (APPENDIX 2A)

ELISA plate (e.g., Immulon 1B or 4HBX; Dynex Technologies)Plastic film (e.g., Nescofilm, Parafilm, Saran wrap)Multichannel pipettor21-G hypodermic needleSide-arm flaskMicrotiter plate reader

Coat ELISA plate with integrin1. Dilute purified integrin to ∼1 µg/ml with DPBS.

At least a 50-fold dilution is required, otherwise the detergent present in the purifiedintegrin interferes with adsorption to the plate.

2. Add the diluted integrin to the wells of an ELISA plate (100 µl/well). Leave a set ofwells empty for measuring binding of the ligand to BSA.

The authors normally perform the assay using 4 to 6 replicates for each sample.

Immulon 1B and 4 HBX ELISA plates (Dynex) are suitable. More recently, the authors havefound that one-half area EIA/RIA plates (Costar) also work well, and have the advantagethat similar results can be obtained with half as much integrin (i.e., 50 �l/well)

3. Wrap the plate in plastic film and store overnight at room temperature.

Alternatively, the plate can be stored for up to 1 week at 4°C.

Block the ELISA plate4. Add 25 µl blocking solution to each integrin-containing well using a multichannel

pipettor, then remove the solution by aspiration using a 21-G hypodermic needleattached by tubing to a side-arm flask connected to a vacuum source, or by invertingthe plate over a sink and flicking out the liquid.

A small amount of blocking solution is added to the wells before aspirating the integrinsolution because the authors have found that this renders the wells hydrophilic and preventsthem from drying out when they are aspirated. Drying out of the wells destroys the activityof some of the integrin.

5. Add 200 µl blocking solution to each well (including those used for testing bindingto BSA alone) using a multichannel pipettor. Leave the plate at room temperature for1 to 3 hr, then aspirate or flick out the blocking solution.

6. Add 200 µl of binding buffer to each well using a multichannel pipettor. Remove thebuffer by aspirating or flicking it out. Repeat two times.

7. Remove residual liquid by inverting the plate and striking it hard several times ontoadsorbent paper towels.

Add biotin-labeled ligand8. Dilute the biotin-labeled ligand in binding buffer to the appropriate concentration.

Add 100 µl of this solution to each experimental well.

The appropriate concentration must be determined by pilot experiments. A concentrationof 0.1 �g/ml works well for biotinylated 80-kD fragment of fibronectin (Mould et al.,1995a).

Other reagents (e.g., mAbs, peptides, or synthetic compounds) can be added simultaneouslywith the ligand at this stage to test for their effects on ligand binding.


9.4.4

AnalyzingIntegrin-

DependentAdhesion

9. Cover the plate with plastic film and incubate 3 hr at 37°C.

A cell culture incubator is suitable for this incubation.

10. Aspirate solutions from the wells to remove unbound ligand.

11. Wash wells three times with 200 µl binding buffer (see step 6). Remove the residualbuffer (see step 7).

Detect bound biotin-labeled ligand12. Dilute ExtrAvidin-peroxidase reagent 1:500 in binding buffer. Add 100 µl of the

diluted ExtrAvidin-peroxidase reagent to each well using a repeating pipettor. Incubatethe plate 10 to 15 min at room temperature. During this time prepare the ABTS reagent.

13. Aspirate solutions from wells to remove unbound ExtrAvidin peroxidase reagent.

14. Wash wells two times with 200 µl of binding buffer and then two times with 400 µlof binding buffer. Remove the residual buffer (see step 7).

15. Add 100 µl of ABTS reagent to each well using a repeating pipettor. Allow thereaction to proceed until a strong (but not dark) green color is obtained (typically 10to 30 min).

16. Stop the reaction by adding 100 µl of 2% SDS solution to each well using a repeatingpipettor.

17. Read the plate using an automatic microtiter plate reader at 405 nm.

18. Calculate the mean and standard deviation of the absorbance readings for ligandbinding to integrin, and for ligand binding to BSA alone, using the followingequations.

SUPPORTPROTOCOL 1

INTEGRIN PURIFICATION

This protocol describes the affinity purification of the integrin α5β1 from human placentausing anti-β1 and anti-α5 mAbs. The procedure can be adapted to purify other integrinsfrom different tissue sources or from pellets of cultured cells. If one is starting fromcultured cells, typically enough cells should be used to give at least 10 ml of packed cells,and the volumes of reagents and resins should be scaled down 2- to 4-fold.

Materials

Human placenta (from maternity unit of local hospital; process within a few hoursof delivery)

Homogenization buffer (see recipe)1% (w/v) Virkon (Merck) in water

net binding to integrin mean of absorbance readings for ligand binding to integrin

mean of absorbance readings for ligand binding to BSA alone

= −( )

( )

standard deviation of net binding

standard deviation of absorbance readings

for ligand binding to integrin standard

deviation of absorbance readings for ligand

binding to BSA alone

=+

(

) (

)

2

2


9.4.5

Cell Adhesion

Extraction buffer (see recipe), 4°CSepharose 4B resin (Sigma)Rat IgG–Sepharose resin (see Support Protocol 2)mAb 13 (anti-β1)–Sepharose and mAb 16 (anti-α5)–Sepharose (see Support

Protocol 2)Wash buffer (see recipe), 4°CElution buffer (see recipe), 4°C1 M Tris⋅Cl, pH 8.2 (APPENDIX 2A; store up to 6 months at 4°C), 4°C0.1 M Tris⋅Cl, pH 8.3 (APPENDIX 2A)/0.1% (w/v) Triton X-100 (Ultra grade, Sigma),

4°C (store up to 3 months at 4°C)Phosphate-buffered saline (PBS; prepare using 10× stock solution from Life

Technologies) containing 0.05% (w/v) sodium azide (add from 20% w/vsodium azide stock in H2O)

PBS (Life Technologies)5× SDS-PAGE sample buffer (see recipe)6% SDS-PAGE gel (UNIT 6.1)

Large scissorsBlenderBeckman J6-B centrifuge with JA-10 and JA-20 rotors (or equivalent refrigerated

centrifuge)500-ml polycarbonate centrifuge bottles (Nalgene)50-ml polyallomer centrifuge tubes (Nalgene)Econo-Pac 20-ml disposable polypropylene columns (Bio-Rad)50-ml screw-top polypropylene tubes (Becton Dickinson Labware)Rotating platform (Cole-Parmer)1.6 × 20–cm C16 column (Amersham Pharmacia Biotech)Peristaltic pumpFraction collector0.8-cm diameter Poly-Prep 2-ml disposable polypropylene column (Bio-Rad)

Additional reagents and equipment for SDS-PAGE and staining of gels (UNIT 6.1)

CAUTION: Human placenta should be treated as potentially biohazardous; take suitableprecautions such as wearing latex gloves, eye protection, and a lab coat. The homogeni-zation should be performed in a primary cell culture cabinet and any spillage of homo-genate or extract should be treated with 1% Virkon. Centrifuge bottles and tubes shouldbe soaked in 1% Virkon after use.

Homogenize placenta1. Cut the placenta (minus umbilical cord and amniotic membranes) into small chunks

using large scissors, and place the pieces in a blender with ∼500 ml of coldhomogenization buffer. Homogenize the placenta using a moderate speed for ∼1 min.Pour the homogenate into 500-ml polycarbonate centrifuge bottles and store at −70°Cuntil required (up to 2 years).

One placenta yields ∼1 liter of homogenate. Commercially available laboratory homoge-nizers may be used, but a robust domestic blender is adequate for this purpose.

Extract the homogenate2. Thaw the homogenate in a cold room (preferable) or at room temperature overnight.

Perform all subsequent operations at 4°C where possible.

3. Centrifuge the homogenate 10 min at 4400 × g (5000 rpm in a JA-10 rotor), 4°C.Discard the supernatant into a bucket containing 5 liters of 1% Virkon, and resuspend


9.4.6

AnalyzingIntegrin-

DependentAdhesion

the pellets in 600 ml of homogenization buffer. Centrifuge again under the sameconditions and discard the supernatant as described.

Most of the soluble proteins have been removed at this stage. The next step uses detergent(Triton X-100) to solubilize proteins (including integrins) from cell membranes. Theextraction buffer contains protease inhibitors and BSA to minimize proteolytic degradationof the integrins.

4. Extract pellet by adding 400 ml extraction buffer to the centrifuge bottle and keepingon ice for ∼1 hr, shaking vigorously every few minutes to ensure that the pellets arefully resuspended.

The extract obtained will be red in color because not all of the hemoglobin has beenremoved in step 3.

5. Centrifuge the extract 10 min at 6400 × g (6000 rpm in a JA-10 rotor), 4°C. Pipet thesupernatant into 50-ml centrifuge tubes, then centrifuge 30 min at 48,000 × g (20,000rpm in a JA-20 rotor), 4°C.

Purify the �1 integrins6. Pack a 20-ml disposable column with 4 ml of Sepharose 4B by pouring 8 ml of a

50% suspension of Sepharose 4B into the column. Preclear and filter the supernatantfrom step 5 by passing it through the column and collecting the flowthrough.

Several columns can be used simultaneously to speed up this step.

7. Mix the flowthrough with 8 ml of a 50% suspension of rat IgG–Sepharose inscrew-top 50-ml polypropylene centrifuge tubes and agitate for 2 hr on a rotatingplatform. Remove the rat IgG–Sepharose by pouring through 20-ml disposablecolumns (filtration is accomplished by the fritted disc at the bottom of the column).

This step removes proteins that bind to rat IgG. If a murine mAb is being used in thesubsequent purification steps mouse IgG–Sepharose should be used in place of ratIgG–Sepharose. The authors normally discard the rat IgG–Sepharose at this stage.

8. Mix the flowthrough from step 7 with 8 ml of mAb 13–Sepharose in 50-ml poly-propylene tubes and agitate for 2 hr on a rotating platform. Recover the mAb13–Sepharose by pouring through a 20-ml disposable column. Retain the flow-through and store at 4°C.

9. Resuspend the mAb 13-Sepharose in wash buffer, pack the suspension into a 1.6 ×20–cm C16 column, and wash the column overnight at ∼10 ml per hr with wash buffer,delivered via a peristaltic pump.

10. Elute integrin by passing elution buffer through the column at 45 ml/hr for 30 min.During this time collect 2-min (1.5-ml) fractions (using a fraction collector) into tubesto which 0.5 ml of 1 M Tris⋅Cl, pH 8.2, has been added. Mix the fractions with thisbuffer as they elute from the column to ensure prompt neutralization. Store fractionsat 4°C.

11. Neutralize the column immediately with 20 ml of 0.1 M Tris⋅Cl (pH 8.3)/0.1% TritonX-100 at a flow rate of 45 ml/hr. Reequilibrate the column with PBS/0.05% sodiumazide and store the mAb 13–Sepharose (removed from the column) at 4°C.

Because of the low pH used to elute the integrin, the mAb 13–Sepharose graduallydeteriorates in its capacity for integrin purification. However, in our experience the columncan be reused about ten times before replacement is necessary.


9.4.7

Cell Adhesion

Analyze the purified �1 integrins12. To 25-µl aliquots of the fractions, add 25 µl 1× PBS and 12.5 µl of 5× SDS-PAGE

sample buffer to each aliquot. Heat the samples in a boiling water bath for 3 min andrun on a 6% SDS-polyacrylamide gel (UNIT 6.1).

13. Stain the gel with Coomassie blue for 1 hr (UNIT 6.1). Destain the gel and check forelution of integrin. A typical elution profile is shown in Figure 9.4.1.

The flowthrough from step 8 can be reapplied to the mAb 13–Sepharose, after anyprecipitate has been removed by centrifuging for 10 min at 4400 × g (5000 rpm in a JA-10rotor), 4°C (follow step 8 onwards; elute and neutralize the column before chroma-tographing the flowthrough a second time, then pool the β1 integrin fractions; repeat untilthe yield of β1 integrins is markedly lower than from the first purification). In the authors’experience, the mAb 13 purification step needs to be repeated several times before all theβ1 integrins have been depleted from the extract.

Purify the integrin �5�114. Pool the fractions from step 10 that contain the purified β1 integrin. Centrifuge the

pooled fractions 10 min at 48,000 × g (20,000 rpm in a JA-20 rotor), 4°C. Mix thesupernatant with 2 ml of mAb 16–Sepharose for 2 hr on a rotating platform. Pack thesuspension into a 0.8-cm diameter 0.8-cm diameter Poly-Prep 2-ml disposablecolumn and wash with 12 ml of wash buffer.

15. Elute α5β1 with 5 ml of elution buffer, added in 0.4-ml aliquots. Collect 0.4-mlfractions in 1.5-ml microcentrifuge tubes containing 0.1 ml of 1 M Tris⋅Cl, pH 8.2.Mix the fractions with this buffer as they elute from column to ensure promptneutralization. Store fractions up to 2 years at −70°C.

16. Neutralize the column with 5 ml of 0.1 M Tris⋅Cl (pH 8.3)/0.1% (w/v) Triton X-100.Re-equilibrate the column with PBS/0.05% sodium azide and store the mAb 16–Sepharose at 4°C.

Analyze the purified �5�117. Analyze 25-µl aliquots of the fractions by SDS-PAGE as described in steps 12 and

13.

- 206

- 105

Figure 9.4.1 SDS-polyacrylamide gel of sequential fractions from low-pH elution of mAb 13column. The major bands observed are (from top) α1 subunit, other α subunits, and β1 subunit. Themigration positions of molecular weight standards are indicated (in kDa).


9.4.8

AnalyzingIntegrin-

DependentAdhesion

The major bands detected by Coomassie blue staining should be those corresponding toexpected positions of the �5 and �1 subunits (Fig. 9.4.2). The molecular weights are ∼150kDa for �5 and ∼130 kDa for �1.

SUPPORTPROTOCOL 2

COUPLING OF ANTIBODIES TO SEPHAROSE

Suitable mAbs for purification may be available in-house or can be generated usingpublished protocols (e.g., Akiyama et al., 1989). Some anti-integrin hybridomas areavailable from cell culture collections (e.g., ATCC). The authors have found that theanti-β1 mAb 13 and the anti-α5 mAb 16 are particularly suitable for affinity purificationof α5β1. Both mAbs are available from K.M. Yamada, National Institutes of Health.

Materials

Antibodies/IgG to be coupled: anti-β1 and anti-α5 mAbs (e.g., mAb 13 and mAb16) and rat IgG (Sigma)

Coupling buffer: 0.5 M NaCl/0.1 M NaHCO3 (store up to 6 months at roomtemperature)

CNBr-activated Sepharose (Sigma)1 mM HCl1 M ethanolamine in H2O (store up to 6 months at room temperature)Acetate wash buffer: 0.1 M sodium acetate, pH 4 (adjust with glacial acetic

acid)/0.5 M NaCl (store up to 6 months at room temperature)Tris wash buffer: 0.1 M Tris⋅Cl, pH 8 (APPENDIX 2A)/0.5 M NaCl (store up to 6

months at room temperature)Phosphate-buffered saline (PBS; prepare using 10× stock solution from Life

Technologies) containing 0.05% (w/v) sodium azide (add from 20% sodiumazide stock solution in H2O)

PBS (Life Technologies)

Buchner funnel with medium-porosity fritted-glass discConical flask with side arm50-ml screw-top polypropylene centrifuge tubesRotating platform (Cole-Parmer)

Additional reagents and equipment for dialysis (APPENDIX 3)

- 237

- 112

Figure 9.4.2 SDS-polyacrylamide gel of sequential fractions from low-pH elution of mAb 16column. The major bands observed are (from top) α5 subunit and β1 subunit. The migration positionsof molecular weight standards are indicated (in kDa).


9.4.9

Cell Adhesion

1. Dialyze the antibodies (if necessary) into an amine-free buffer (e.g., PBS or couplingbuffer). Measure the absorbance of an aliquot of the dialyzed mAb at 280 nm. Diluteor concentrate antibodies to ∼1 mg/ml

Dialysis is only necessary where the mAb is supplied in amine-containing buffer (e.g., Tris).

2. Pre-swell the CNBr-activated Sepharose in ∼50 ml of 1 mM HCl. Wash with ∼200ml of 1 mM HCl per ml of resin in a Buchner funnel. Prepare a 50% resin slurry inPBS.

3 g of CNBr-activated Sepharose swells to ∼10 ml.

3. Mix the antibodies (diluted to ∼1 mg/ml) with CNBr-activated Sepharose as follows.

a. For mAb 13: ∼2 mg mAb per ml Sepharose slurry

b. For mAb 16: ∼5 mg mAb per ml Sepharose slurry

c. For rat IgG: 2 mg IgG per ml Sepharose slurry.

Allow coupling reaction to proceed 2 hr at room temperature.

4. Following the coupling reaction, add the mixture to a Buchner funnel attached to aconical flask with side arm and remove the supernatant by vacuum filtration.Determine the absorbance at 280 nm for an aliquot of the recovered supernatant andcompare to the starting value according to the following equation, where D is thedilution factor (total volume divided by volume of starting solution).

Typical coupling efficiencies are 95% to 99%.

5. Wash the Sepharose in the Buchner funnel with 20 ml of coupling buffer and removeall buffer under suction. Remove the Sepharose from the funnel using a spatula, placeit in a 50-ml conical polypropylene centrifuge tube with 20 ml of 1 M ethanolamine,and incubate 1 hr at room temperature on a rotating platform

The buffer for washing may be added in several aliquots if the capacity of the funnel is low.

6. Return the Sepharose to the Buchner funnel and wash with three alternating applica-tions, 50 ml each, of acetate wash buffer and Tris wash buffer.

7. Wash the Sepharose with 20 ml of PBS and store in PBS/0.05% sodium azide at 4°C.

The buffer for washing may be added in several aliquots if the capacity of the funnel is low.

SUPPORTPROTOCOL 3

BIOTINYLATION OF INTEGRIN LIGANDS

This protocol describes labeling of ligands for in vitro integrin-binding assays (see BasicProtocol 2). Biotin is covalently conjugated to amino groups on the ligand to permitquantification of bound ligand.

Materials

Ligand of interestCoupling buffer 0.5 M NaCl/0.1 M NaHCO3 (store up to 6 months at room

temperature)Sulfo-NHS Biotin (Pierce)Tris/saline: 25 mM Tris⋅Cl (pH 7.4)/150 mM NaCl

coupling efficiencyof filtrate

of starting solution= − ×�

��

��×1 100%280

280

A D

A


9.4.10

AnalyzingIntegrin-

DependentAdhesion

Tris/saline containing 0.05% sodium azideRotating platform (Cole-Parmer)

Additional reagents and equipment for dialysis (APPENDIX 3) and protein assay(APPENDIX 3)

1. Dialyze ligand into 1 liter of coupling buffer (APPENDIX 3) for at least 2 hr at roomtemperature.

About 0.5 ml of ligand at a concentration of ∼0.5 mg/ml gives sufficient material for a largenumber of assays.

2. Add an equal mass of sulfo-NHS-biotin (∼0.25 mg) to the dialysate in a 1.5-mlmicrocentrifuge tube and mix on a rotating platform 30 min at room temperature.

For some proteins, sulfo-NHS-LC-biotin (Pierce) gives higher signals than sulfo-NHS-bio-tin in the solid phase assay.

3. Dialyze the solution against two changes of 1 liter Tris/saline, and once against 1 literof Tris/saline, 0.05% azide (at least 2 hr per dialysis) at room temperature.

4. Microcentrifuge the dialysate for 15 min in a 1.5-ml microcentrifuge tube at maxi-mum speed, room temperature.

This removes any large aggregates or precipitate from the solution.

5. Measure the concentration of biotinylated protein in the supernatant using, forexample, the BCA assay (APPENDIX 3). Store up to 6 months at 4°C.

Alternatively, many biotinylated proteins can be stored in aliquots at −70°C. This procedurewill typically provide sufficient material for >100 α5β1 fibronectin assays. How muchmaterial will be required for any assay will depend upon the affinity of the integrin-ligandinteraction.

REAGENTS AND SOLUTIONS

Use deionized or distilled water in all recipes and protocol steps. For common stock solutions, seeAPPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

ABTS reagentABTS buffer:0.05 M Na2HPO4

0.1 M sodium acetateAdjust pH to 5.0 using concentrated HClStore up to 3 months at room temperatureABTS reagent: Immediately before assay, dissolve 11 mg of 2,2′-azinobis(3-ethyl-benzthiazoline)sulfonic acid (ABTS; Sigma) in 0.5 ml water. Mix 67 µl of 30%H2O2 with 7 ml water. Add 0.5 ml of this ABTS solution to 10 ml ABTS buffer (seeabove) and 100 µl H2O2 solution. Mix thoroughly.

This amount of reagent is sufficient for one full 96-well plate assay.

Binding bufferMix the following components in the order indicated:150 mM NaCl25 mM Tris⋅Cl, pH 7.4 (APPENDIX 2A)1 mM MnCl2

0.1% (w/v) BSA (fraction V; Sigma, 99% pure)Prepare fresh

This is conveniently prepared from Tris/saline [25 mM Tris⋅Cl (pH 7.4)/150 mM NaCl] anda stock solution of 1 M MnCl2. BSA is then added.


9.4.11

Cell Adhesion

Blocking solutionPrepare a solution of 25 mM Tris⋅Cl containing 150 mM NaCl. Add sufficient 20%sodium azide stock for a final concentration of 0.05% (w/v). Add BSA (fraction V;Sigma, 98% pure) for a final concentration of 5% (w/v) and dissolve by vigorousstirring. Centrifuge the solution in 50-ml centrifuge tubes for 5 min at 2800 × g(4000 rpm in a JA-10 rotor), and filter the supernatant through a 20-ml disposablecolumn. Store up to 3 months at 4°C.

This is conveniently prepared from Tris-saline [25 mM Tris⋅Cl (pH 7.4)/150 mM NaCl] towhich sodium azide is added from a 20% stock solution. BSA is then added and dissolvedby vigorous stirring. The solution is then centrifuged and filtered as above. Final concentra-tions are 150 mM NaCl; 25 mM Tris⋅Cl, pH 7.4, 5% (w/v) BSA, and 0.05% (w/v) sodiumazide.

Elution buffer10 mM sodium acetate, pH 3.251 mM CaCl2

1 mM MgCl2

0.1% (w/v) Triton X-100 (Sigma, Ultra grade)Store up to 1 month at 4°C

Extraction buffer150 mM NaCl25 mM Tris⋅Cl, pH 7.4 (APPENDIX 2A)2% (w/v) Triton X-100 (Sigma, Ultra grade)1 mM PMSF (100 mM stock solution prepared in isopropanol; also see APPEN-

DIX 1B)10 µg/ml leupeptin (1 mg/ml stock solution prepared in water; also see APPEN-

DIX 1B)2 mg/ml BSA (fraction V; Sigma, 98% pure)Prepare fresh, then cool on ice

Homogenization buffer150 mM NaCl25 mM Tris⋅Cl, pH 7.4 (APPENDIX 2A)0.005% (w/v) digitoninStore for up to 3 months at 4°C

SDS-PAGE sample buffer, 5×25% (v/v) glycerol125 mM Tris⋅Cl, pH 6.8 (APPENDIX 2A)10% (w/v) SDS0.1% (w/v) bromophenol blueStore indefinitely at 4°C. Warm in a hot water bath and mix well before use.

Wash buffer150 mM NaCl25 mM Tris⋅Cl, pH 7.4 (APPENDIX 2A)1 mM CaCl2 (add from 1 M stock)1 mM MgCl2 (add from 1 M stock)0.1% (w/v) Triton X-100 (Sigma, Ultra grade)Store up to 1 month at 4°C

This is conveniently prepared from Tris-saline [25 mM Tris⋅Cl (pH 7.4)/150 mM NaCl] andstock solutions of 1 M CaCl2 and 1 M MgCl2.


9.4.12

AnalyzingIntegrin-

DependentAdhesion

COMMENTARY

Background InformationMonoclonal antibodies were of crucial im-

portance in the initial identification of cell sur-face receptors that mediated adhesion of cellsto extracellular matrix components (e.g.,Wayner and Carter, 1987). A wide range of wellcharacterized anti-integrin mAbs are now com-mercially available for use in cell attachmentand spreading assays. The choice of whichanti-integrin mAbs to use is determined in partby the substrate. For example, if collagen typeI is the substrate, anti-α1 and anti-α2 mAbsshould be tested, as these are the likely integrinsinvolved. The complement of integrins ex-pressed by the cell also helps to determinewhich mAbs should be tested. If the profile ofintegrin expression is unknown, it can be deter-mined by flow cytometry or by immunoprecipi-tation of surface-labeled cells. Conversely, if amAb blocks cell adhesion, it is important todemonstrate that the corresponding integrinsubunit is expressed by the cells.

Historically, the first purification of an inte-grin receptor was a major advance in under-standing the molecular basis of cell-matrix in-teractions (Pytela et al., 1985). This and latermethods employed a ligand affinity column asthe major purification procedure. In particular,fibronectin fragments have been used to purifyα5β1, and RGD peptides have been used topurify αVβ3 and αIIbβ3 (Pytela et al., 1987;Smith and Cheresh, 1988; Yamada and Yamada,1990). Ligand affinity columns remain themethod of choice where mAbs are unavailable.However, the use of mAbs has the advantagethat the purification is more specific (e.g., sev-eral different integrins bind to fibronectin af-finity columns) and higher yields of integrinscan be obtained. Multiple β1 integrins can alsobe obtained from the same source. For example,other β1 integrins from human placenta that arepresent in the flowthrough from the mAb 16column can be purified using other specificanti-α mAbs. For some assays, and particularlywhere one β1 integrin predominates in thetissue or cell extract, it may be sufficient topurify the total β1 integrins and use this par-tially purified preparation in the solid-phaseassay. For example, β1 integrins purified fromMOLT-4 cells (Newham et al., 1998) contain∼75% α4β1, the remainder being α5β1. Theprotocol described here can be adapted to pu-rify other integrins from other tissues or frompellets of cultured cells.

The major advantage of assays using puri-fied integrins, as compared to cell-based assays,is that integrin-ligand binding can be studied inisolation. Integrin clustering, signaling, and cy-toskeletal interactions are all known to affectthe strength of adhesion in cell-based assays.Furthermore, adhesion may be modulated byindirect effects (e.g., by signaling from othercell-surface receptors). Although doubts areoften expressed as to whether plastic-adsorbedintegrin is representative of integrin in its nativeenvironment, a number of careful studies haveshown no significant differences between thebehavior of integrins in solid-phase assays andon cell surfaces. Hence, this approach has beenbroadly validated. The first solid-phase inte-grin-ligand binding assay was described byCharo et al. (1991) for studying fibrinogenbinding to αIIbβ3. The α5β1-fibronectin assaydeveloped by the author of this unit is bothextremely sensitive and highly versatile. Forexample, the author has described how theassay can be used to investigate the effects onligand binding of divalent cations, activatingand inhibitory mAbs, peptide inhibitors, andmutations (Mould et al., 1995a,b, 1996, 1997).Another important area in which this type ofassay is finding use is in the pharmacologicalscreening of inhibitors of integrin-ligand inter-actions. This assay can give information aboutthe inhibitory potency of a compound andwhether it is a direct competitive or allostericinhibitor of ligand binding. The attenuation ofmAb epitopes can also provide data on thelocation of the binding site of an inhibitor onthe integrin (Mould et al., 1997).

The author’s preferred method for labelingof integrin ligands is biotinylation, because ofits safety and simplicity. One potential draw-back is that if one or more lysyl residues in theligand are crucial for integrin binding, theirmodification may render the ligand inactive. Inthis case, a possible solution may be to reducethe amount of biotinylation reagent so thatsome of the lysyl residues remain unmodified.Other labeling methods such as radioiodinationcan also be used. Alternatively, if the ligand isa recombinant protein, a “tag” such as an epi-tope sequence or the Fc region of IgG can beincorporated for use in the detection of boundligand.


9.4.13

Cell Adhesion

Critical Parameters andTroubleshooting

For cell-based assays, as described in UNIT

9.1, the health of the cells and careful prepara-tion are important for achieving optimalspreading or attachment. In order to optimizethe sensitivity of cell attachment or spreadingto inhibition, the author recommends that aconcentration of substrate be chosen that gives50% to 70% of maximal adhesion (unless thisvalue is low, in which case a concentration ofsubstrate that gives near maximal adhesionshould be used). If the level of adhesion is nearmaximal, inhibitors are less effective at block-ing adhesion.

Failure of a mAb to inhibit spreading orattachment can normally be taken to mean thatthe integrin it recognizes is not involved inadhesion. However, it is important to check thatthe antibody is effective in a system whereinhibition should be observed (e.g., an anti-α5mAb should block spreading of HT-1080 fi-brosarcoma cells on fibronectin). Conversely,if an antibody does inhibit, it is important totest that it does not inhibit adhesion to aninappropriate ligand (e.g., an anti-α5 mAbshould not perturb HT-1080 cell spreading oncollagen). Most of the antibodies described inTable 9.4.1 are well characterized and shouldnot show any non-specific effects. However, asdescribed earlier, it is essential that the antibod-ies not contain sodium azide.

It is also important to bear in mind thatinhibition of adhesion can be caused by “cross-talk”—i.e., where ligation of one integrin (e.g.,by peptide) indirectly affects the activity of a

second integrin via intracellular signaling(Diaz-Gonzalez et al., 1996). While it is oftendifficult to rule out such effects, they normallyonly cause a partial reduction in adhesion,whereas adhesion is frequently totally ablatedby specific antibody or peptide inhibition. Fi-nally, if anti-integrin mAbs or peptides do notcompletely block cell adhesion, it is possiblethat non-integrin receptors may play some role.This may be observed particularly in cell at-tachment assays. For example, cell-surface pro-teoglycans contribute to melanoma cell attach-ment to the heparin-binding domain of fibro-nectin (Mould et al., 1994).

For solid-phase assays, the specificity of theassay must be tested carefully. The most impor-tant test for specificity is the ability of unlabeledligand to compete with labeled ligand for bind-ing to the integrin. Hence, in the presence of alarge excess of unlabeled ligand, very littlebinding of labeled ligand should be observed.Nearly all integrin-ligand interactions are diva-lent-cation dependent. Hence, replacing theMn2+ in the binding buffer with EDTA shouldreduce binding to levels similar to that observedfor BSA-coated wells. Further tests for speci-ficity can be carried out. For example: (1) mAbsthat are inhibitory in cell-based assays shouldalso inhibit ligand binding in solid-phase as-says, (2) mutations known to affect integrinbinding sites should perturb ligand recognition,and (3) known ligand mimetics (e.g., RGDpeptides for α5β1 or CS1 peptide for α4β1)should block ligand binding. All of these testshave been performed for the α5β1-fibronectin

Table 9.4.2 Troubleshooting Guide for Problems Encountered in Solid-Phase Assays

Problem Possible cause Solution

High background binding to BSA-coated wells

Insufficient blocking of wellsLigand concentration too high

Block for longer time (e.g., overnight)Test range of ligand concentrations foroptimal signal/background

Spuriously high signal in somewells

Insufficient washingTops of wells contaminated

Follow washing protocol carefullyAdd reagents to the center of wells

Wide variation in signal inexperimental wells

Integrin added to plate insufficientlymixedWells aspirated before adding blockingreagentPlate contaminated, e.g., by dust

Mix diluted integrin thoroughly beforecoating the plateAdd blocking reagent before removingcoating solutionUse fresh, clean plates

Low signal above backgroundbinding to BSA

Insufficient integrinLigand concentration too lowInactive ligand

Use lower dilution of integrinUse higher concentration of ligandCheck activity of ligand in cell-basedassay


9.4.14

AnalyzingIntegrin-

DependentAdhesion

assay. Table 9.4.2 is a troubleshooting guide forsolid-phase ligand-binding assays.

For integrin purification, it is essential tohave sufficient tissue or cell pellets for theextraction and enough mAb-Sepharose to per-form a successful purification. Table 9.4.3 is atroubleshooting guide for integrin purification.

Anticipated ResultsFor cell-based assays, in most cases, it is

possible to obtain levels of spreading or attach-ment of >50%. It should be feasible usinganti-integrin mAbs, either alone or in combina-tion, to reduce the level of spreading or attach-ment to close to that seen on BSA.

For integrin purification, yields of α5β1 are∼2 mg per placenta (estimated by Coomassieblue staining). Each full 96-well-plate solid-phase assay uses <10 µg of integrin. Hence, thisis enough to perform a large number (∼200) ofsolid-phase assays. In these assays, a strongpositive signal (OD405 ∼1) is normally achievedin ≤30 min, whereas background binding toBSA is generally very low (OD405 ∼0.1).

Time ConsiderationsCell attachment and spreading assays are

quick to perform. The actual assays can becarried out in about half a day or less; however,particularly if many different mAbs or peptidesare being tested, quantification of spreadingassays can take several hours.

Purification of α5β1 from placenta takes ∼3days in total. Allow an additional day for prepa-ration of buffers and mAb-Sepharose. Thesolid-phase assay can be performed in 5 to 8 hrbut only requires 1 to 2 hr of hands-on time.However, the assay needs to be planned inadvance so that the appropriate number of wellscan be coated with integrin overnight. Plates

can be coated with integrin several days inadvance.

Literature CitedAkiyama, S.K., Yamada, S., Chen, W.-T., and Yama-

da, K. 1989. Analysis of fibronectin receptorfunction with monoclonal antibodies: Roles incell adhesion, migration, matrix assembly, andcytoskeletal organization. J. Cell Biol. 109:863-875.

Charo, I.F., Nannizzi, L., Phillips, D.R., Hsu, M.A.,and Scarborough, R.M. 1991. Inhibition of fi-brinogen binding to GP IIb-IIIa by a GP IIIapeptide. J. Biol. Chem. 266:1415-1421.

Diaz-Gonzalez, F., Forsyth, J., Steiner, B. andGinsberg, M.H. 1996. Trans-dominant inhibi-tion of integrin function. Mol. Biol. Cell7:11939-11951.

Mould, A.P., Wheldon, LA., Komoriya, A., Wayner,E.A., Yamada, K., and Humphries, M.J. 1990.Affinity chromatographic isolation of the mela-noma adhesion receptor for the IIICS region offibronectin and its identification as the integrinα4β1. J. Biol. Chem. 265: 4020-4024.

Mould, A.P., Askari, J.A., Craig, S.E., Garratt, A.N.,Clements, J., and Humphries, M.J. 1994. Inte-grin α4β1-mediated melanoma cell adhesionand migration on vascular cell adhesion mole-cule-1 (VCAM-1) and the alternatively splicedIIICS region of fibronectin. J. Biol. Chem.269:27224-27230.

Mould, A.P., Akiyama, S.K., and Humphries, M. J.1995a. Regulation of integrin α5β1-fibronectininteractions by divalent cations: Evidence fordistinct classes of binding sites for Mn2+, Mg2+,and Ca2+. J. Biol Chem. 270:26270-26277.

Mould, A.P., Garratt, A.N., Askari, J.A., Akiyama,S.K., and Humphries, M.J. 1995b. Identificationof a novel monoclonal antibody that recognisesa ligand-induced binding site epitope on the β1subunit. FEBS Lett. 363:118-122.

Mould, A.P., Akiyama, S.K., and Humphries, M.J.1996. The inhibitory anti-β1 integrin mono-clonal antibody 13 recognises an epitope that isattenuated by ligand occupancy: evidence for

Table 9.4.3 Troubleshooting Guide for Problems Encountered in Integrin Purification

Problem Possible cause Solution

Large number of proteins copurifywith integrins

Inadequate preclearing or filtration ofextract, or precipitate forms duringpurification procedures

Recentrifuge after preclearing andfiltering extract on Sepharose 4B, orwhen any precipitate is visible

Small amounts of integrinspurified

Insufficient mAb coupled to SepharoseAffinity of mAb too lowColumn has been used many times

Couple more mAb to SepharoseUse mAb with higher affinityReplace with fresh mAb-Sepharose

Integrin degraded Insufficient levels of protease inhibitorsin extraction bufferExtraction or other manipulationsperformed at too high a temperature

Increase levels of protease inhibitorsand BSA in extraction bufferPerform all manipulations at 4°C or onice


9.4.15

Cell Adhesion

allosteric inhibition of integrin function. J. Biol.Chem. 271:20365-20374.

Mould, A.P., Askari, J.A., Aota, S., Yamada, K., Irie,A., Takada, Y., Mardon, H.J., and Humphries,M.J. 1997. Defining the topology of integrinα5β1-fibronectin interactions using inhibitoryanti-α5 and anti-β1 monoclonal antibodies: Evi-dence that the synergy sequence of fibronectin isrecognised by the amino-terminal repeats of theα5 subunit. J. Biol. Chem. 272:17283-17292.

Newham, P., Craig, S.E., Clark, K., Mould, A.P., andHumphries, M.J. 1998. Analysis of ligand-in-duced and ligand-attenuated epitopes on the leu-kocyte integrin α4β1: VCAM-1, MAdCAM-1and fibronectin induce distinct conformationalchanges. J. Immunol. 160:4508-4517.

Pytela, R., Pierschbacher, M.D., and Ruoslahti, E.1985. Identification and isolation of a 140 kd cellsurface glycoprotein with properties expected ofa fibronectin receptor. Cell 40:191-198.

Pytela, R., Pierschbacher, M.D., Argraves, S.,Suzuki, S., and Ruoslahti, E. 1987. Arginine-gly-cine-aspartic acid adhesion receptors. MethodsEnzymol. 144:475-489.

Smith, J.W. and Cheresh, D.A. 1988. The Arg-Gly-Asp binding domain of the vitronectin receptor.J. Biol. Chem. 263:18726-18731.

Wayner, E.A. and Carter, W.G. 1987. Identificationof multiple cell adhesion receptors for collagenand fibronectin in human fibrosarcoma cells pos-sessing unique α and common β subunits. J. CellBiol. 105:1873-1884.

Yamada, K.M. and Yamada, S.S. 1990. Isolation offibronectin receptors. In Receptor Purification(G. Litwack, ed.) pp 435-449. Humana Press.Clifton, NJ.

Contributed by A. Paul MouldUniversity of ManchesterManchester, United Kingdom


9.4.16

AnalyzingIntegrin-

DependentAdhesion

UNIT 9.5Analysis of Cell-Cell Contact Mediated by IgSuperfamily Cell Adhesion Molecules

The calcium-independent cell adhesion molecules (CAMs) constitute a large family ofcell surface molecules. A major group among these are the immunoglobulin superfamily(IgSF) molecules. IgSF-CAMs may be composed of immunoglobulin (Ig) folds only, Igfolds linked to fibronectin type III (FnIII) folds, or Ig folds linked to protein modulesother than FnIII folds. The IgSF is a large protein superfamily comprising >100 proteinsinvolved in cell-cell adhesion. Its members are found in vertebrates, invertebrates, andalso in yeast. Most of the molecules of the IgSF are cell surface molecules that aremembrane-anchored either by a single transmembrane segment or by a glycosylphospha-tidylinositol (GPI) anchor that is posttranslationally attached to the C-terminus. Some ofthe IgSF-CAMs also occur in soluble form, e.g., in the cerebrospinal fluid or the vitreousfluid of the eye, due to a cleavage of the GPI-anchor or the membrane-proximal peptidesegment. In some cases, such as NCAM, various forms may be generated by alternativesplicing.

This unit provides protocols for the purification of IgSF-CAMs from tissue extracts andtissue culture supernatants and for the analysis of the adhesive functions of IgSF-CAMswith isolated molecules and in the cellular context. Following personal expertise, theauthors have added a few frequently used functional assays demonstrating the role ofIgSF-CAMs in neural development, such as neurite outgrowth from cultured neurons,and the use of antibodies for the inhibition of IgSF-CAM functions in vitro. The firstgroup of protocols describe affinity purification of IgSF-CAMs (see Basic Protocol 1),preparation of the affinity column (see Support Protocol 1), solubilization of membraneproteins (see Support Protocol 2), transient transfection of HEK 293 cells to expressIgSF-CAMs (see Support Protocol 3), and detection of IgSF-CAMs by dot blot analysis(see Support Protocol 4). Assays using fluorescent microspheres with coupled proteinsare used for one type of functional analysis based either on interactions between micro-spheres (see Basic Protocol 2) or on interactions between microspheres and cultured cells(see Basic Protocol 3). There are two protocols for coupling proteins to microspheres:coupling proteins to fluorescent microspheres (see Support Protocol 5) and couplingproteins to glutaraldehyde-activated amino beads (see Support Protocol 6). A secondgroup of protocols analyze the functions in cell-based assays. Trans-interactions arestudied using IgSF-CAM-transfected myeloma cells (see Basic Protocol 4). This protocolrequires stable transfection of myeloma cells (see Support Protocol 7). Cis-interactionsare detected by chemical cross-linking (see Basic Protocol 5) and antibody co-capping(see Basic Protocol 6). IgSF-CAMs and other substrates have the ability to promoteneurite outgrowth (see Basic Protocol 7), which requires coating of the growth surfacewith IgSF-CAM (see Support Protocol 8), nitrocellulose as a binder for the substrate ofinterest (see Support Protocol 9), poly-D-lysine (see Support Protocol 10), collagen (seeSupport Protocol 11), or laminin (see Support Protocol 12). Differential fixation protocolsare used for fluorescent immunohistochemistry samples (see Support Protocol 13) or formorphological analyses (see Support Protocol 14). Finally, there is a protocol forassessing the effect of inhibiting CAM-CAM interactions in vitro (see Basic Protocol 8).

Supplement 11

Contributed by Peter Sonderegger, Stefan Kunz, Christoph Rader, Daniel M. Suter, and Esther T. StoeckliCurrent Protocols in Cell Biology (2001) 9.5.1-9.5.52Copyright © 2001 by John Wiley & Sons, Inc.

9.5.1

Cell Adhesion

PREPARATION OF IgSF-CAMs

BASICPROTOCOL 1

Purification of IgSF-CAMs by Immunoaffinity Chromatography

The best method for the purification of native, functionally intact proteins is certainly theuse of standard chromatography, such as ion exchange, hydrophobic interaction, and gelpermeation columns. However, the establishment of such a standard purification protocolcan be time-consuming and requires expensive equipment. As the specific characteristicsexploited for chromatography differ from protein to protein, purification protocols cannotbe generalized. A faster and less expensive way to purify IgSF-CAMs is to use affinitychromatography. Tissue homogenates, body fluids in the case of secreted proteins, or celllines engineered to express a particular protein of interest, either transiently or stably, canbe used as a source for IgSF-CAM purification. Affinity chromatography makes use ofthe specific binding properties of the proteins, e.g., receptors for their ligands, enzymesfor their substrates, or antibodies for their antigens. For IgSF-CAMs, the purification byimmunoaffinity is most commonly used. A disadvantage of affinity purification is thepossibility of the loss of activity, as the protein is sometimes eluted from the column byrather harsh conditions. The protocol described below has been successfully used for thepurification of functionally intact IgSF-CAMs (Stoeckli et al., 1991, 1996; Rader et al.,1993).

The general principle of immunoaffinity chromatography is the use of a resin-coupledmonoclonal antibody directed against the protein to be purified. Generally, activatedSepharose resins are used. The resin is packed into a column connected to a peristalticpump and to a UV-detector to monitor the elution profile of the column. The purity of theeluted protein is analyzed by SDS-PAGE (UNIT 6.1). Here, we describe a purificationprotocol for a membrane-bound IgSF from brain membranes (see Support Protocol 2 formembrane preparation and protein solubilization).

Materials

CNBr-activated Sepharose 4B column (see Support Protocol 1)Loading buffer: 0.5% CHAPS in PBS with Ca2+/Mg2+

Elution buffer: 0.5% CHAPS in 50 mM diethylamineProtein solution (see Support Protocol 2)1 M Tris⋅Cl, pH 7.0 (APPENDIX 2A)PBS with Ca2+/Mg2+ (see recipe)0.02% (v/v) merthiolate or equivalent bacteriostatic agent

Prepare column1. Rinse the column extensively with loading buffer, especially for a column that was

prepared earlier and has been stored for a while. Add 2 to 3 vol (i.e., 3 times thevolume of the column) of elution buffer to the column to test the stability of thecolumn under the elution conditions and to make sure that the column does not containany contaminations, such as unspecifically bound proteins from previous use of thecolumn.

2. Properly reequilibrate the column to loading conditions before loading the proteinsolution (see Support Protocol 2 for protein solution preparation).

As the binding affinity of antibodies is usually not temperature-sensitive, the authorsrecommend running the affinity column at 4°C rather than at room temperature. Keepingthe column and the protein solution to be loaded at 4°C helps to prevent contaminationand slows down degradation of the proteins.


9.5.2

Cell-Cell Contactby Ig Superfamily

Cell AdhesionMolecules

Load the column3. During loading of the protein solution onto the column, adjust the flow rate to a

sufficiently slow rate (usually 0.1 ml/min is chosen for good yields) to allow goodinteraction between antigen and antibodies coupled to the sepharose beads.

The capacity of the column can be tested by collecting fractions of the flow-through andsubsequent immunoblot analysis (see Support Protocol 4). For best yield, the capacity ofthe column should not be exhausted. Typically, column volumes of 1 ml are used.

Elute column4. Incubate the column in 0.9 vol elution buffer for 10 min.

This will increase the elution efficiency (a higher concentration of the eluted protein ineluate, rather than a broad elution peak with a long tail). Do not expose the column toelution conditions for longer times than necessary, because the high pH of the buffer couldbe detrimental for the affinity column.

5. Elute the column at a rate of 1 ml/min. Collect the eluate in vials containing enough1 M Tris⋅Cl, pH 7.0, to buffer the eluate at a neutral pH value.

For a 1-ml column, 1-ml fractions are collected in 1.5-ml microcentrifuge tubes.

The volume of 1 M Tris⋅Cl, pH 7.0, required for restoring the pH should be determined at4°C, as the pH value of Tris is extremely temperature sensitive.

The flow rate for elution can be much higher than that for loading. However, check themaximal flow rate acceptable for a specific resin. For Sepharose resins a maximal flowrate of 30 ml hr−1cm−2 is recommended.

The eluate can be stored a few days at 4°C; for longer storage below −20°C is recom-mended. However, keep in mind that repeated thawing and freezing is detrimental to theprotein. Furthermore, freezing of dilute protein solutions is not recommended.

6. Re-equilibrate the column to loading conditions for a second run, or prepare thecolumn for storage.

Regenerate and store the affinity column7. Immediately after elution re-equilibrate the column to neutral pH values with PBS

with Ca2+/Mg2+. For storage, add 0.02% merthiolate or an equivalent bacteriostaticagent to the PBS to prevent bacterial growth. Store the column at 4°C.

Prevent drying of the column during storage. The column can be stored for several monthsat 4°C.

SUPPORTPROTOCOL 1

Preparation of the Affinity Column

This protocol only describes the preparation of an immunoaffinity column. Generally,because IgSF-CAMs have low binding affinities for their binding partners and have noenzymatic activity that could be used for substrate-based purification, the use of immu-noaffinity columns is the method of choice.

However, a prerequisite is the availability of a monoclonal antibody against the proteinto be purified. This antibody is covalently coupled to a Sepharose resin. Affinity columnsare versatile, they can be used for tissue homogenates, solubilized membrane proteins, orculture supernatants from cell lines that are engineered to produce and release IgSF-CAMs. If stored appropriately, affinity columns can be reused many times over severalmonths.


9.5.3

Cell Adhesion

Materials

CNBr-activated Sepharose 4B gel1 mM HClBuffer I: 0.5 M NaCl in 0.1 M NaHCO3, pH 8.3Monoclonal antibody against the protein to be purified0.2 M glycine, pH 8.0Buffer II: 0.5 M NaCl in 0.1 M sodium acetate, pH 4.0Loading buffer (see Basic Protocol 1)

Sintered glass filter connected to a vacuum pumpColumn (e.g., Poly-Prep column, Bio-Rad)U-bottomed polypropylene vial that can be closed tightly

1. Soak 1:2 (w/v) CNBr-activated sepharose 4B in 1 mM HCl for 15 min at roomtemperature.

For a 1-ml column, start with 350 to 400 mg Sepharose.

2. Transfer beads to a sintered glass filter connected to a vacuum pump, and wash with≥25 vol of 1 mM HCl, followed by buffer I.

It is very important to prevent the beads from drying between the additions of buffer (onegram dry resin yields ∼3.5 ml swollen gel).

3. Transfer the slurry to a U-bottomed polypropylene vial containing the antibodysolution in buffer I. Carry out the reaction for 2 hr at room temperature. Close thevial tightly.

The final concentrations should be: 100 mg Sepharose (dry weight) and 5 mg antibody perml coupling reaction mix.

Ideally, rotating the vial end-over-head is used to maximize the coupling efficiency.

Do not use a magnetic stirrer, which will damage the agarose beads.

4. Stop the reaction by gently centrifuging the Sepharose beads for 5 min at 2000 × g,room temperature.

5. Add 3 vol 0.2 M glycine, pH 8.0, to the pellet and continue to rotate the vialend-over-head for an additional 2 hr.

Collect the supernatant of the coupling reaction to check the coupling efficiency.

6. Pack the slurry into a column.

Typically, column volumes are ∼1 ml with a column diameter of 0.5 cm.

7. Wash the column with 5 vol buffer I followed by 5 vol buffer II. Repeat washprocedure four times to remove excess uncoupled ligand.

8. Before loading the protein solution (see Support Protocol 2), wash the columnthoroughly with 25 to 30 vol loading buffer.

An affinity column can be used repeatedly, if stored appropriately with an antibacterialagent (e.g., 0.02% merthiolate) at 4°C. Before using the column after storage, rinse thecolumn extensively with loading buffer. Use 2 to 3 vol elution buffer to clean the column,restore loading conditions by rinsing thoroughly with loading buffer (≥10 vol).


9.5.4



SUPPORTPROTOCOL 2

Solubilization of Membrane Proteins

Most IgSF-CAMs are either glycosyl-phosphatidylinositol-anchored or transmembraneproteins, therefore, they have to be solubilized from cell membranes with detergents. Inthe authors’ experience, the use of CHAPS has given the best results with respect to yieldand functional integrity of the purified proteins. Keep in mind that for many functionalassays, detergent removal from the protein solution is necessary after purification; thepresence of detergents can also interfere with binding assays. Especially sensitive assaysare those that involve neurons, such as neurite outgrowth assays (see Basic Protocol 7).For removal of detergents from protein solutions, the authors have used SM-2 beads fromBioRad or Calbiosorb from Calbiochem. The use of high concentrations of detergentscan interfere with the purification by immunoaffinity columns, therefore, dilution of theprotein solution after the solubilization step, i.e., before the solution is loaded onto theaffinity column, is recommended. The protocol given below has been successfully usedfor the purification of functionally intact L1/NgCAM from E14 chicken brain membranes(e.g., Stoeckli et al., 1991, 1996). It is adapted from the purification protocol describedby Grumet and Edelman (1984). However, the authors have used the same protocol forthe solubilization and purification of other IgSF-CAMs (Rader et al., 1993; Fitzli et al.,2000). For storage, membranes and proteins can be frozen at the indicated steps. However,repeated freez-thaw cycles are detrimental for proteins and for high yields of intactproteins and should be minimized.

NOTE: All protocols using live animals must first be reviewed and approved by anInstitutional Animal Care and Use Committee (IACUC) or must conform to governmentalregulations regarding the care and use of laboratory animals.

Materials

14-day-old chicken embryo brains, freshly frozen in liquid nitrogenLiquid nitrogenCa2+/Mg2+-free buffer (CMF buffer; see recipe)0.8 M and 2.25 M sucrose in PBS1 M and 2 M NaCl in PBS50 mM triethylamine0.5% and 1% CHAPS in PBS

Mortar and pestleDounce homogenizerCentrifuge tubes for Sorval SS-34 or equivalent rotor38-ml polycarbonate tubes for ultra high-speed centrifuge

Prepare membranes1. Remove brains of 14-day-old chicken embryos and immediately freeze in liquid

nitrogen.

If necessary, brains can be stored at −70°C for extended periods of time.

2. Cool a mortar and pestle of sufficient size with liquid nitrogen and grind frozen brainsin batches to a fine powder. Add small volumes of liquid nitrogen to keep brains/brainpowder frozen during grinding.

Carefully avoid thawing of the tissue at any time.

3. Add ∼3 vol CMF buffer to 1 vol brain powder. Homogenize the brain powder in aDounce homogenizer.

4. Centrifuge homogenate for 20 min at 45,000 × g (10,000 rpm in a SS-34 rotor), 4°C.


9.5.5

Cell Adhesion

5. Resuspend the pellets in 1.5 to 2 vol of 2.25 M sucrose, use Dounce homogenizer toget homogenous suspension.

6. Transfer ∼24 ml suspension to each polycarbonate ultracentrifuge tube, overlay with1⁄4 vol of 0.8 M sucrose.

7. Centrifuge for 60 min at 150,000 × g, 4°C.

8. Transfer the membranes that are accumulated in the interphase to the Douncehomogenizer, resuspend them in 25 vol PBS.


10. Decant supernatant and resuspend in PBS, centrifuge as in step 9, and resuspendpellets in smallest possible volume of PBS for storage at −20°C, or use directly forstripping.

Samples can be stored up to 1 month at −20°C, or at −70°C for longer storage.

Strip packed membranes11. Add 1 vol of 2 M NaCl in PBS to packed membrane suspension (from step 10),

homogenize suspension.

12. Add 1 M NaCl in PBS to a final volume of 4 to 5 times the volume of the packedmembranes.

13. Stir suspension 1 hr on a magnetic stirrer at low speed, 4°C, to strip membranes fromperipheral membrane proteins.


15. Resuspend pellets in 20 vol of 50 mM triethylamine, stir for 60 min at 4°C.

16. Repeat centrifugation step 14. Carefully remove the supernatant with a pipet, as themembranes do not form a stable pellet after the high pH extraction step.

17. Resuspend the pellets in PBS.


19. Repeat washing the membranes with PBS at least one time to restore a pH valuebetween 7.2 and 7.6. Keep an aliquot for measuring the protein concentration.

20. Freeze the stripped membranes at −20°C or use directly for solubilization step.

For storage longer than 1 month, store at −70°C.

Solubilize integral membrane proteins21. If frozen membranes are used, they should be washed once again with PBS (steps 17

and 18).

22. Transfer pellets into a Dounce homogenizer and resuspend in 1 vol of 1% CHAPSbuffer. Add 4 vol of 0.5% CHAPS buffer.

For good solubilization the protein concentration should be adjusted to ∼1 mg/ml.

23. Extract the membrane proteins by stirring the suspension for 60 min at 4°C.

24. Centrifuge for 60 min at 150,000 × g , 4°C.

25. Combine supernatants, measure the volume, and remove aliquot to determine theprotein concentration.

Do not freeze the solubilized proteins, but use directly for affinity purification step.


9.5.6



SUPPORTPROTOCOL 3

Production of Recombinant CAM by Transient Transfection of HEK293 Cellswith Calcium Phosphate

Transient transfection of HEK 293 cells with expression vectors containing cDNAs ofIgSF-CAMs represents a fast and convenient method for the production of intermediateamounts (100 µg to 1 mg) of recombinant protein. The human embryonic kidney cell line293 (HEK 293) is a well established, easily transfectable cell line that is widely used forthe expression of recombinant proteins. The transfection method of choice with HEK 293cells is always calcium phosphate transfection, an inexpensive, convenient technique thatresults in high efficiencies of transfection.

DNA can be introduced into a wide variety of cultured cell lines as a calcium phosphatecomplex (Graham and van der Eb, 1973; Wigler et al., 1977). The transfected DNA caneither integrate into the genome of the recipient cell, resulting in stable transgeneexpression accompanied by a stably altered phenotype of the cell (stable transfection) orremain episomal resulting in only transient expression of the transgene (transient trans-fection). The following protocol can be used likewise for the generation of stable cell linesby subsequent selection for stable transfectants or for transient expression only.

Efficient transfection requires the formation of a fine precipitate of calcium phosphate inthe presence of DNA. The formation of the DNA-containing calcium phosphate particlesis initiated under defined chemical conditions, in the absence of cells or serum. Theparticle size is the most critical parameter regarding efficiency of transfection, that isuptake of DNA-containing calcium phosphate particles by the cells. The main determi-nants of particle size are calcium and phosphate concentrations, the concentration ofDNA, size of DNA fragments involved, pH, temperature, and time of incubation. Afterinitial formation of the DNA-containing calcium phosphate particles, the precipitate isadded to the cells. During the incubation of the precipitate with the cells, the formationof DNA-containing calcium phosphate particles continues and preexisting particles growin size. The particles adhere to the cells and are taken up by endocytosis. After a few hoursof exposure, the medium is changed and the cells start to express the recombinant protein.The period of efficient transgene expression varies between different expression vectorsbut lasts generally for a few days.

Materials

HEK 293 cellsCell culture medium for HEK 293 cells (see recipe)Purified DNA of interestCaCl2 solution (see recipe)HBS solution (see recipe)175-cm2 tissue culture flasksAdditional reagents and equipment for trypsinizing cells (UNIT 1.1)

NOTE: All solutions and equipment coming into contact with living cells must be sterile,and aseptic technique should be used accordingly.

NOTE: All cell culture incubations should be carried out in a 37°C, 5% CO2 humidifiedincubator.

1. For transient transfection, grow HEK 293 cells of low passage number (passage <40)and split cells every 2 to 3 days before 80% confluency is reached.

By frequent splitting and growth in subconfluent density, the cells remain in their exponen-tial growth phase and are highly susceptible for transfection up to about passage number40.


9.5.7

Cell Adhesion

2. One day prior to the experiment, trypsinize cells briefly (UNIT 1.1) and prepare a singlecell suspension. Plate 5 × 106 cells in 30 ml of medium per 175-cm2 flask and incubatefor 16 to 20 hr at 37°C, 5% CO2 incubator.

For optimal transfection efficiency, 60% to 70% confluency of cells is required. This allowstwo additional cell divisions after transfection. Some 293 lines adhere only poor to tissueculture plastic, especially after transfection. To achieve more stable adherence, tissueculture flasks can be coated with poly-D-lysine (see Support Protocol 10).

HEK 293 cells do not need a medium change prior to addition of the calcium phosphateprecipitate.

3. Mix purified DNA (40 to 80 µg in a maximum volume of 100 µl) with 1.5 ml CaCl2

solution and incubate for a few minutes at room temperature.

For efficient formation of the DNA-containing calcium phosphate particles, temperature iscritical. The solutions should be kept at room temperature for a couple of hours before use.The optimal amount of DNA depends on the particular expression construct used fortransient transfection and has to be determined individually for each system.

4. Add 1.5 ml of HBS solution and mix well. Incubate for 1 to 15 min at roomtemperature and then add the reaction mixture to the cells (from step 2).

The formation of the DNA-containing calcium phosphate particles starts immediately aftermixing the two solutions and can be observed by a slightly opaque appearance of thesolution. Longer incubation periods generally result in the formation of a precipitate withlarger particles. The optimum time of incubation critically depends on the size of theplasmids used.

The growth rate of the calcium phosphate particles frequently correlates inversely with thesize of the plasmids. For very large plasmids(>30 kb), preincubation of no longer than 1min is recommended.

5. Incubate the DNA-containing calcium phosphate particles with the cells for 4 to 6 hrat 37°C in an incubator.

The formation of the calcium phosphate precipitate can be monitored under an invertedlight microscope with a 63× objective. The precipitate should become visible as tinyparticles (∼300 nm in diameter) especially on the surface between the cells. However, longexposure to the atmosphere outside of the incubator should be avoided.

6. Change medium after the incubation period and continue incubation. Begin testingfor expression of the recombinant protein 1 day after transfection.

The cells should now express the recombinant protein for a few days.

Uptake of the DNA-containing calcium phosphate particles by cells can be checked byexamination of free surfaces between cells. The margins surrounding the cells should becleared of particles, due to their uptake by the cells.

SUPPORTPROTOCOL 4

Detection of IgSF-CAMs by the Dot Immunoblot Method

A rapid and convenient method for the detection of IgSF-CAMs in eluates from chroma-tography or affinity columns are dot immunoblots. Similar to immunoblots, dot blots area semi-quantitative method to detect proteins transferred onto a nitrocellulose membranevia a chromogenic reaction. For this purpose, secondary antibodies coupled to peroxidaseor alkaline phosphatase are used to detect binding of the first antibodies directed againstthe protein of interest. However, in contrast to immunoblots, proteins are not separatedon a polyacrylamide gel, therefore dot blots are a much faster way to demonstrate thepresence of a specific protein in a given sample. Furthermore, as the proteins are directlyapplied to the nitrocellulose membrane for dot blots, there is no loss due to difficulties in


9.5.8



transfer from the gel to the membrane. The fact that only small volumes can be appliedonto the membrane for dot blots may, however, limit its use for detection of proteins indilute solutions.

The procedure described here can also be used to stain IgSF-CAMs after immunoblotting.Use manufacturer’s manuals and protocols for sample preparation, SDS-PAGE, andblotting of proteins onto nitrocellulose (also see UNITS 6.1 and 6.2).

MaterialsProtein solution of interestTBS: 0.2 M NaCl in 50 mM Tris, pH 7.4Blocking solution: 2% (w/v) milk powder in TBS with or without 0.1% (w/v)

Tween 20Antibody against the protein of interest diluted in blocking solutionSecondary antibody coupled with horseradish peroxidase (HRP) diluted in

blocking solution4-chloro-1-naphthalene solution (see recipe)

0.2-µm nitrocellulose membrane (e.g., Schleicher and Schuell)96-well platesRotary shaker

1. Place matching round disks of 0.2-µm nitrocellulose membrane into wells of a96-well plate.

When large numbers of samples have to be analyzed, the use of a commercially availableprotein-dispersing device is advised (e.g., Dot Punch IM-96, Inotech AG). These devicesallow the application of protein solutions to the membrane before they are put into thewells. This is more convenient and less time-consuming compared to the proceduredescribed below.

2. Apply small volumes (<10 µl) of the protein solution onto the membrane.

The solution should not cover the entire membrane but remain confined to a segment of themembrane (dot). This is important in order to ensure the attachment of sufficient amountsof proteins per area to reach detection limits. If the protein solution is very dilute, the dottingprocedure can be repeated after drying the nitrocellulose after the first application. Therepeated application of protein to the membrane increases the amount of the protein ofinterest.

3. Dry the membranes to optimize protein adherence to the nitrocellulose.

4. Wash membranes three times with 100 µl TBS per well.

5. Block protein adsorbance of the membrane by incubating ≥30 min with blockingsolution, room temperature.

Generally, 2% to 5% milk powder in TBS with or without 0.1% Tween 20 works well as ablocking solution. In some cases, addition of 2% to 10% (v/v) serum in TBS improves thesignal-to-background ratio.

6. Apply 1 to 10 µg/ml first antibody diluted in blocking solution and shake for ≥1 to 2hr, room temperature, on a rotary shaker.

If necessary, incubate overnight.

Shaking the 96-well plate improves antibody/antigen interaction.

7. Wash thoroughly with TBS (≥3 times).

8. Incubate with blocking solution for ≥30 min before applying the secondary antibody.


9.5.9

Cell Adhesion

9. Apply 1 to 10 µg/ml of secondary antibody coupled with horseradish peroxidasediluted in blocking solution to the membrane and incubate 1 hr at room temperature.

As a secondary antibody, the use of a peroxidase-coupled antibody is described, but goodresults can also be obtained with phosphatase-coupled antibodies. Generally, an antibodyconcentration of 1 to 10 µg/ml is used to get good signal-to-noise ratios but batches orproducts from different suppliers vary.

10. Remove antibody solution and wash membrane thoroughly with at least three changesof TBS.

11. Visualize the antibody by adding 0.1 ml of 4-chloro-1-naphthalene solution.

Generally, the dark blue color develops immediately.

12. Stop the reaction by rinsing the membranes with water.

FUNCTIONAL ASSAYS WITH PURIFIED PROTEINS

BASICPROTOCOL 2

Analysis of Protein Interactions with Fluorescent Microspheres

There are essentially two ways to analyze the binding properties of cell adhesionmolecules, (1) binding assays based on purified proteins, and (2) binding assays based onheterologously expressed proteins in their physiological environment (cell aggregationassays). This protocol describes a method that utilizes biochemically purified proteinscoupled to fluorescent microspheres, whereas Basic Protocol 4 is based on cell aggrega-tion assays. If pure cell adhesion molecules are available, proteins can be coupled topolystyrene beads of different fluorescent colors. Coupling can be either by covalentlinkage or by hydrophobic adsorption of the protein to the microsphere surface. Afterdispersion of the beads by ultrasonication, pairwise combinations of differently coupledbeads aggregate during a particular incubation period. Binding activities are analyzed bythe evaluation of aggregate formation both qualitatively, by inspection on a fluorescentmicroscope, and quantitatively, using a fluorescence-activated flow cytometer. Althoughthis protocol is a reliable method to detect binding activities of IgSF-CAMs and has beenused by several laboratories, one has to consider false negative results as well asdiscrepancies between results of bead and cell aggregation assays. Some discrepancieshave been explained by differences in the orientation of proteins coupled to beads incomparison to proteins on the surface of cells.

Materials

Protein-conjugated fluorescent microspheres; stock solutions contain 1011 beads/mlin 0.5% (w/v) BSA (see Support Protocol 5; Duke Scientific; BangsLaboratories; Polysciences)

0.5% (w/v) BSA solution (see recipe)0.5 mg/ml Fab fragments of antibodies against proteins of interest in PBS

(optional)0.5% (w/v) trypsin (optional)PBS (see recipe)

Water bath sonicator (Branson Ultrasonics)Fluorescence microscope equipped with FITC and TRITC filters0.5-ml microcentrifuge tubeRotatorGlass microscope slidesRefrigerated microcentrifugeFluorescence-activated flow cytometer


9.5.10



Prepare mixtures of fluorescent microsphere1. Sonicate test tubes containing stock solutions of protein-conjugated microspheres in a

water bath sonicator for 2 min at room temperature. Use lowest sonicator output settingneeded to achieve monodisperse bead solution to prevent protein structure damage.

Make relatively small aliquots of protein-conjugated bead stocks because repetitive ultra-sonication may damage protein structure.

For the applications described here, fluorescent microspheres with a nominal diameter of0.5 µm work best. Both covalent coupling of proteins via surface-exposed functional groupsor hydrophobic adsorption may be used. The best mode of coupling may depend on theparticular nature of the protein to be studied.

The authors have consistently used Covaspheres fluorescent microspheres from DukeScientific (Kuhn et al., 1991; Suter et al., 1995; Rader et al., 1996; Kunz et al., 1998; Fitzliet al., 2000). Fluorescent Covaspheres with a nominal diameter of 0.5 µm were easy tohandle, allowed covalent coupling of the proteins, and were readily detectable in the lightmicroscope and cell sorter. However, Duke Scientific has discontinued the distribution ofCovaspheres, but a new line of beads with well characterized fluorescence and chargedensity is available. Different functional groups for covalent coupling of proteins will beoffered, including carboxylate and aldehyde groups.

Support Protocol 5 describes covalent coupling of proteins to Covaspheres as well as thehydrophobic adsorption of proteins to polystyrene beads. With some modifications, it mayalso be suited for other types of beads.

The authors have added a support protocol that can be used for coupling proteins tomicrospheres exhibiting amino groups on their surfaces (see Support Protocol 6).

2. Check monodispersity of bead stock solution by quick inspection of a 1:100 dilutionin 0.5% BSA under a fluorescence microscope.

3. Immediately after ultrasonication, prepare different test mixtures by combiningprotein-coupled beads of different fluorescent colors. Add 2 µl of green-fluorescentbeads conjugated with protein 1 and 2 µl of red-fluorescent beads conjugated withprotein 2 into a 0.5-ml microcentrifuge tube and bring to a final volume of 20 µl with0.5% BSA solution (the concentration of each species of beads in the test mixture is1010/ml). Vortex beads.

Use as many controls as possible. Typical control incubations are:

a. Each type of beads alone.

b. Beads conjugated with control proteins that should not bind to protein of interest(e.g., BSA, non-immune IgG).

c. Protein-conjugated beads that were either incubated in boiling water for 10 min ortreated with 0.5% trypsin overnight.

These treatments should abolish specific protein interactions.

d. Pretreat beads with Fab fragments that are specific to the conjugated protein ofinterest. For this purpose, incubate an appropriate volume of the bead stock solutionwith Fab from polyclonal IgG at a concentration of 0.5 mg/ml in PBS for 2 hr at roomtemperature. Remove unbound antibodies by three consecutive washes with PBS.For solution changes, centrifuge beads in a microcentrifuge tube for 10 min at 16,000× g, 4°C. Dissolve possible aggregates of beads by ultrasonication as in step 1.

4. Incubate by slowly rotating test mixtures protected from light for 1 hr at roomtemperature on a rotator.

If such a rotator is not available, invert tubes a few times every 15 min. Tubes can be lightprotected with aluminum foil.


9.5.11

Cell Adhesion

Analyze by fluorescence microscopy5. Analyze the test mixtures for aggregate formation by fluorescence microscopy.

Prepare a 1:10 dilution of the different incubations in 0.5% BSA solution and place20 µl of this dilution on a glass slide for inspection.

Most fluorescent microspheres emit a relatively intense signal. Therefore, antifading agentsare not necessary for inspection. After a while, aggregates will settle and pictures can betaken for documentation or quantification of aggregate sizes. Adhesive interactions be-tween two different proteins coupled to beads labeled with FITC and TRITC, respectively,should result in aggregates consisting of up to 100 beads of both colors that are equallydistributed throughout the clusters. Carefully check for bleed-through between the differentfluorescence channels.

Analyze by fluorescence-activated flow cytometry6. Use flow cytometric analysis for quantification of aggregate formation. Calibrate the

flow cytometer correctly for nonaggregated beads of each color.

7. Dilute test mixtures 1:1000 in 0.5% BSA solution and inject into a fluorescence-ac-tivated flow cytometer equipped with appropriate FITC and TRITC filter sets (Kuhnet al., 1991).

Always use fluorescence microscopy in parallel to flow cytometer analysis to obtaininformation on the distribution of the beads in aggregates (see Critical Parameters andTroubleshooting).

8. Use uncoated, nonaggregating beads to determine relative fluorescence intensity(RFI) of individual beads of each color. Compensate for the spectral overlap of theFITC and TRITC emission electronically.

9. Record the data output for number, size, and composition of mixed aggregates.

Data can be presented as two-dimensional contour or dot plots with the RFIs in the FITCand TRITC channel as x- and y-coordinates indicating size and composition of aggregates(Kuhn et al., 1991; Suter et al., 1995). The signal intensity represents the number ofaggregates of a certain size and composition. The RFI values of single beads will allow theinvestigator to set the boundaries between mixed aggregates and aggregates that consistonly of one species of beads. Determination of these boundaries is necessary to calculatethe percentage of beads found in mixed aggregates as an indicator for protein binding.

SUPPORTPROTOCOL 5

Coupling Proteins to Fluorescent Microspheres

This support protocol describes a method for the preparation of protein-conjugatedfluorescent microspheres required for the protein interaction studies described in BasicProtocols 2 and 3. It is based on the procedure that the authors have used for the couplingof proteins to Covaspheres (Duke Scientific). However, the same procedure has success-fully been used for the adsorption of proteins to polystyrene beads, which bind proteinsby hydrophobic interactions.

Additional Materials (also see Basic Protocol 2)

1011 unconjugated beads/ml fluorescent polystyrene microspheres, 0.5-µmdiameter (Duke Scientific; Bangs Laboratories; Polysciences)

Biochemically purified IgSF-CAMs (see Basic Protocol 1) in a phosphate-basedbuffer system (either PBS or 20 mM sodium phosphate, pH 7.0)

1. Sonicate tubes containing stock solutions of 1011 unconjugated beads/ml fluorescentpolystyrene microspheres in a water bath sonicator for 2 min at room temperature.


9.5.12



2. If bead solution is monodisperse, transfer 100 µl of beads to a new tube and add 50µg of biochemically purified IgSF-CAMs in PBS to a total volume of 1 ml (1010

beads/ml during coupling). Vortex tubes.

Before coupling, test protein preparations carefully for purity with SDS-PAGE to avoidfalse positive, as well as false negative, results.

Store proteins under sterile conditions in PBS or 20 mM sodium phosphate, pH 7.0, 4°C.For highly stable proteins, storage for up to 1 year at 4°C under sterile conditions ispossible.

Cell adhesion molecules with transmembrane and cytoplasmic domains (e.g., NgCAM andNrCAM) require the presence of detergents such as CHAPS and deoxycholate duringpurification. The authors found that biological activity, such as binding, is better preservedwhen these detergents are maintained during protein storage at 4°C and during couplingto beads.

3. Incubate the mixture for 1 hr in a 37°C water bath. Mix periodically by inverting tube.

4. Centrifuge for 10 min at 16,000 × g, 4°C. Remove supernatant containing unboundprotein and save for coupling analysis. Resuspend beads in 1 ml of 0.5% BSAsolution.

This step is necessary to block residual binding sites that could result in unspecificbead-bead interactions.

5. Sonicate beads as in step 1 and incubate 30 min at room temperature.

6. Centrifuge beads as in step 4 and resuspend pellet in 100 µl of 0.5% BSA solution.Store beads at 4°C.

Protein-conjugated microspheres should never be frozen and can be kept up to 1 year at4°C (depending on protein stability).

7. (Optional) Determine coupling yield by SDS-PAGE of serial dilutions of proteinsamples taken before and after coupling.

Densitometric analysis of bands after silver staining or immunoblot detection allows thedetermination of both coupling efficiency and the number of protein molecules bound permicrosphere.

Typically, a coupling yield of >50% is obtained under the conditions described. In the caseof the IgSF-CAMs axonin-1 and NgCAM, ∼16,000 molecules were found to bind perCovasphere bead (Kuhn et al., 1991).

SUPPORTPROTOCOL 6

Covalent Coupling of Proteins to Glutaraldehyde-Activated Amino Beads

As an alternative method for coupling proteins to beads, this protocol describes theglutaraldehyde-activated coupling of proteins to microspheres with amino-functionalgroups. Purified IgSF-CAMs can be coupled directly (not oriented) to amino beads.Alternatively, Fc-containing recombinant proteins can be bound to protein A–conjugatedmicrospheres in an oriented manner.

Additional Materials (also see Basic Protocol 2)

Amino-functional microspheres (e.g., silica aminopropyl beads from BangsLaboratories)

0.1 M NaOH or HCl (optional)8% (v/v) EM-grade glutaraldehyde, newly opened bottle400 µg/ml biochemically purified IgSF-CAMs (see Basic Protocol 1) in a

phosphate-based buffer system (either PBS or 20 mM sodium phosphate, pH7.0)

Blocking solution (see recipe)


9.5.13

Cell Adhesion

1. Prepare an appropriate volume (e.g., 0.5 ml) of 1% amino-functional microspherestock solution in ultra pure water. Wash beads two times with 1 ml water. Centrifugefor 4 min at 16,000 × g, room temperature, to separate microspheres from washsolution. Remove supernatant and resuspend microspheres in 0.5 ml water.

The amino-functional microspheres are available as powder or 10% (w/v) solution. Keepmicrosphere stock solutions at 4°C and never freeze.

2. Before adding the glutaraldehyde solution, check under the microscope whether thebead solution is monodisperse. If the bead solution contains significant clumps,sonicate in the water bath sonicator for 5 min at room temperature. If clumps persist,use a Branson microtip sonicator at the lowest output power needed to disrupt theclumps. Check pH of bead solution with cut pH strips and, if necessary, adjust pH to6.5 or 7.0 with 0.1 M NaOH or HCl, respectively.

Glutaraldehyde activation works best at pH 6 to 7.

3. If the bead solution is monodisperse and has a pH of 6.5 to 7.0, add an equal volumeof a newly opened bottle of 8% EM-grade glutaraldehyde and mix. Incubate beadson rotator for ≥6 hr or overnight at room temperature.

4. Centrifuge beads as in step 1, remove supernatant, and wash activated beads at leastthree times with 1 ml water, and once with the buffer in which the protein is dissolved(PBS or 20 mM sodium phosphate).

5. Add 0.5 ml of 400 µg/ml purified IgSF protein in PBS, pH 7.3, or in 20 mM sodiumphosphate, pH 7.0, to bead pellet and resuspend beads in protein solution (1% beadsduring coupling reaction). Incubate on rotator for 4 hr at room temperature orovernight at 4°C.

If less protein is available, scale down amounts of protein and beads proportionally.

6. Centrifuge beads 4 min at 16,000 × g, room temperature, save supernatant forcoupling analysis on SDS-PAGE, and resuspend beads in 0.5 ml blocking solution.Incubate beads in blocking solution for 30 min at room temperature on rotator.

7. Centrifuge beads again. Resuspend beads in 0.5 ml blocking solution (1% bead stock)and store at 4°C for months.

BASICPROTOCOL 3

Binding of Protein-Conjugated Microspheres to Cultured Cells

This protocol determines binding specificity of a cell adhesion molecule to a receptor ona particular cell type. Both primary cell cultures (Kuhn et al., 1991; Suter et al., 1995)and cell lines (Buchstaller et al., 1996; Rader et al., 1996) can be incubated withprotein-conjugated microspheres under live conditions to determine interactions betweenspecific proteins and cells. Antibody preincubations can be used as a control but also forreceptor identification. Furthermore, cells transiently transfected with mutated receptorforms can be used for the identification of extracellular protein domains necessary forbinding of the cell adhesion protein (Rader et al., 1996; Kunz et al., 1998; Fitzli et al.,2000).

Materials

Primary cultures of neuronal and/or glial cells or other cellsComplete medium used for cell culturesSerum-free BSA-containing cell culture medium1011 beads/ml protein-conjugated fluorescent polystyrene microspheres; stock

solutions in 0.5% BSA (see Support Protocol 5)


9.5.14



0.5 mg/ml Fab against protein of interest in serum-free medium (optional)Fixation solution (see recipe)PBS (see recipe)Mounting medium (see recipe)

Waterbath sonicator37°C, 10% CO2 humidified incubatorGlass microscope slidesFluorescence microscope

Prepare and bind microspheres to cells1. Cultivate primary neurons and/or glial cells in conditions that allow live cell incuba-

tions in relatively small volumes.

For example, grow cells on substrate-coated coverslips using a removable donut-shapedTeflon ring to limit the incubation volume to 200 to 250 �l (Suter et al., 1995).

If culture conditions include the use of serum, prepare also a corresponding serum-free,BSA-containing medium as in Stoeckli et al., 1991; see recipe.

2. Wash cultured cells one time with complete culture medium and one time withserum-free, BSA-containing cell culture medium.

Medium exchanges are carried out carefully with a pipet.

3. Prepare 1:1000 dilutions of 1011 beads/ml protein-conjugated fluorescent polystyrenemicrospheres in serum-free cell culture medium, sonicate dilutions for 2 min, roomtemperature and immediately add to cultured cells.

4. (Optional) To test whether a specific CAM binds to a particular cell type via acharacterized receptor (to which antibodies are available), preincubate cells with 0.5mg/ml Fab fragments in serum-free medium, before adding the beads, for 2 hr in a37°C, 10% CO2 humidified incubator. Remove unbound antibodies by washing twotimes with serum-free medium before proceeding to step 5. To test for the specificityof bead binding to cells, use control protein–conjugated beads as well as beads thatwere preincubated with the corresponding Fab fragments.

5. Incubate the cells with the bead solution (step 3) for 1 hr in a 37°C, 10% CO2

humidified cell culture incubator.

6. Use a Pasteur pipet to carefully aspirate medium with unbound beads. Immediatelyadd serum-free medium. Repeat this step three times.

Do not allow the cells to dry.

Beads bound to live cells can now be inspected. However, if more time is needed foranalysis, the authors recommend fixing the cells. Fixation also allows processing of thecells for immunostaining of marker proteins or CAMs and, therefore, correlative analysisof CAM binding and CAM expression.

Fix cells7. Fix the cells by adding 80 µl of 4× fixation solution to the cultures, which are in 240

µl of serum free medium. Gently mix and incubate 1 hr at 37°C.

8. Wash fixed cells three times with PBS.

9. For immunofluorescence staining of the cells, proceed as described in UNIT 4.3.Otherwise mount cells on a glass microscope slide in mounting medium for inspec-tion under a fluorescence microscope.

Beads are generally intensely fluorescent. Therefore, relatively short exposure times aresufficient when taking pictures.


9.5.15

Cell Adhesion

CELL-BASED ASSAYS FOR IgSF-CAM FUNCTION

BASICPROTOCOL 4

Trans-Interaction Assay with Myeloma Cells

Interactions between IgSF-CAMs that have been determined either by a fluorescentmicrosphere assay or by binding of fluorescent microspheres to cells do not provide anyinformation on whether the interaction occurs between molecules located on differentcells (trans-interaction) or between molecules residing on the membrane of the same cell(cis-interaction). In order to distinguish between a trans- and a cis-interaction, theIgSF-CAMs need to be studied in a natural environment, i.e., as membrane-boundmolecules residing in their proper orientation in a biological membrane. Expression innonadherent myeloma cells (see Support Protocol 7) provides a means of assessingIgSF-CAMs for trans-interactions. Two populations of stably transfected myeloma cellclones are stained with optically distinct intracellular fluorescent dyes. The cells aredissociated, incubated, and examined under a microscope in order to determine whetherre-aggregation has occurred.

To evaluate whether two populations of myeloma cell clones expressing the IgSF-CAMsof interest on their surface can adhere to each other, they are labeled, mixed, dissociated,and allowed to re-aggregate. Labeling with optically distinct fluorescent dyes is necessaryto distinguish the two populations.

Materials

Two populations of myeloma cell clones expressing the CAMs of interestSelection medium, e.g., 5 mM L-histidinol in DMEM supplemented with 10%

(v/v) FCSPBS with Ca2+/Mg2+ (see recipe)Stock solution of green fluorogenic dye, e.g., 1 mM

2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester(Molecular Probes) in DMSO

Stock solution of red fluorogenic dye, e.g., 7.5 mM5-(and-6)-carboxynaphtofluorescein diacetate (Molecular Probes) in DMSO

1% (v/v) FCS in PBS with Ca2+/Mg2+

5 mg/ml p-phenylenediamine in 1% FCS in PBS with Ca2+/Mg2+

15-ml conical polypropylene centrifuge tubesHemacytometerV-shaped 96-well microtiter plate (e.g., Costar, Corning)Centrifuge and rotor for microtiter plates22-G needle attached to 1-ml syringeGlass microscope slideFluorescence microscope with appropriate filters for green and red fluorescence,

e.g., FITC and Texas RedAdditional reagents and equipment for counting cells (UNIT 1.1)

Prepare cells1. Grow transfected myeloma cell clones that express the CAM(s) of interest on their

surface in selection medium to a cell density of ∼5 × 105 cells/ml. Use a hemacytome-ter to monitor the cell density (the aggregation assay requires 5 × 105 cells perpopulation per sample; see UNIT 1.1).

Transfected myeloma cell clones that express high concentrations of a homophilicallytrans-interacting cell adhesion molecule can grow in very large cell aggregates consistingof thousands of cells that are easily visible by eye. The authors found that cultures ofaggregating myeloma cell clones contain more dead cells than nonaggregating myelomacell clones. This might, at least in part, be due to a limited oxygen and nutrition supply to


9.5.16



the center of the aggregate. In order to limit the number of dead cells, aggregating myelomacell clones should be split more frequently avoiding large cell aggregates. The cell densitycan be determined with a hemacytometer after dissociating the cells by repeated pipettingthrough a 22-G needle.

2. Transfer 5 to 10 ml of cell culture into 15-ml conical polypropylene tubes andcentrifuge for 3 min at 500 × g, room temperature. Aspirate the supernatant andresuspend the cells in 1 to 2 ml PBS with Ca2+/Mg2+. Determine the cell density witha hemacytometer (UNIT 1.1). Add PBS with Ca2+/Mg2+ to give a cell density of 5 × 105

cells/150 µl. Pipet 150 µl/well to a V-shaped 96-well plate.

The aggregation assay requires two populations in distinct wells per sample. Use wells A1and A2 for the first sample, B1 and B2 for the next sample, etc.

When desired, the cells can be pre-incubated with antibodies at this step by incubating thecells with Fab monoclonal or polyclonal antibodies in a concentration range of 10 to 500µg/ml in 1% FCS in PBS with Ca2+/Mg2+ for 1 hr at room temperature.

3. Centrifuge the microtiter plate for 2 min at 500 × g, room temperature, remove thesupernatant by flicking plate into a sink (cells will remain in wells). Resuspend cellsin 90 µl PBS with Ca2+/Mg2+.

Label cells4. Prepare fresh working solutions of both, green and red, fluorogenic dyes by diluting

10 µl of the stock solution in 990 µl PBS with Ca2+/Mg2+. Add 10 µl of the appropriateworking solution to the well with the appropriate 90-µl cell suspension. Use onecolumn for one fluorogenic dye (e.g., stain A1, B1, etc. with green and A2, B2, etc.with red). Incubate 30 min at 37°C.

The end concentration of the fluorogenic dyes is 1 µM of 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester and 7.5 µM of 5-(and-6)-carboxynaphtofluo-rescein diacetate. The electrically neutral ester substrates freely diffuse through the cellmembrane into the cell, where they are cleaved into fluorescent products by nonspecificintracellular esterases. The charged fluorescent products are retained by cells with intactplasma membranes. Serum of the cell culture medium contains esterases and has to bewashed away prior to the incubation and avoided during the incubation.

5. Centrifuge microtiter plate and remove the supernatant as in step 3. Resuspend thecells in 150 µl PBS with Ca2+/Mg2+. Repeat step.

6. Centrifuge microtiter plate 2 min at 500 × g, room temperature and remove thesupernatant by flicking plate into sink. Resuspend cells in 75 µl of 1% FCS in PBSwith Ca2+/Mg2+.

Dissociate cells and allow re-aggregation7. Combine complementarily stained cells of one sample in one well (e.g., add A2 to A1,

B2 to B1, etc.). Dissociate cells by slowly pipetting up and down ten times through a22-G needle attached to a 1-ml syringe. Avoid foaming. Incubate 45 min at 4°C.

During re-association the plate should not be moved.

8. Centrifuge microtiter plate for 2 min at 500 × g, room temperature, and remove thesupernatant by flicking plate into sink. Resuspend the cells in 150 µl of 1% FCS inPBS with Ca2+/Mg2+. Repeat step.

9. Centrifuge microtiter plate and remove the supernatant as in step 8. Resuspend thecells in 40 µl of 1% FCS in PBS with Ca2+/Mg2+.


9.5.17

Cell Adhesion

Analyze cells10. Immediately prior to microscopic analysis of an individual sample, add 10 µl of 5

mg/ml p-phenylenediamine in 1% FCS in PBS with Ca2+/Mg2+. Pipet the sampleseveral times up and down using a 200-µl pipet tip. Mount 10 µl of the sample on aglass slide.

The use of the antifading reagent p-phenylenediamine in an end concentration of 1 mg/mlmarkedly preserves the fluorescence intensity.

11. Analyze the aggregates with a fluorescence microscope using FITC and Texas Redfilters.

Filters that allow the simultaneous detection of green and red fluorescence facilitate theanalysis.

SUPPORTPROTOCOL 7

Stable Transfection of Myeloma Cells by Protoplast Fusion

This protocol describes the stable transfection of myeloma cells with a vector that allowsthe surface expression of IgSF-CAMs. A vector particularly suited for myeloma cellexpression was described by Traunecker et al. (1991). Expression by this vector is drivenby an Ig κ promoter and enhancer. The 3′ end of the transcript of interest is spliced ontoan exon encoding the Ig κ constant domain in order to mimic stable Ig transcripts. Thevector contains a histidinol dehydrogenase gene that allows the selection of stabletransfectants in the presence of L-histidinol. L-histidinol is a precursor of L-histamine andan inhibitor of protein synthesis. The vector has been stably transfected into the mousemyeloma cell line J558L for the production of soluble lymphocyte-derived cell-surfacereceptor proteins (Traunecker et al., 1991). The system has also been used for the surfaceexpression of IgSF-CAMs (Rader et al., 1993; Buchstaller et al., 1996; Fitzli et al., 2000).Alternatively, other mammalian expression vectors and myeloma cells can be used.

Myeloma cell clones that stably express large amounts of IgSF-CAMs on their surfacewere generated by a transfection method known as protoplast fusion. Transfection byprotoplast fusion is a highly efficient method for the direct transfer of mammalianexpression vectors from bacteria to mammalian cells (Schaffner, 1980; Sandri-Goldin etal., 1981; Rassoulzadegan et al., 1982; Gillies et al., 1983). It involves digesting bacterialcell walls with lysozyme to produce protoplasts and then fusing the protoplasts tomammalian cells in the presence of polyethylene glycol. The following protocol is basedon the myeloma expression system described by Traunecker et al. (1991) and can easilybe adapted to other systems.

Materials

Glycerol stock of an E. coli strain 803 clone (ATCC #35581) transformed with amammalian expression vector containing the cDNA of the IgSF-CAM ofinterest (store at −80°C)

LB agar/ampicillin plates (see recipe; store at 4°C)DMEM supplemented with 10% (v/v) FCSLB medium (see recipe), prewarmed to 37°C50 mg/ml ampicillin (store at −20°C)60 mg/ml chloramphenicol in ethanol (store at −20°C)DMEM supplemented with 10% (w/v) sucrose and 10 mM MgCl2, prewarm20% (w/v) sucrose in 50 mM Tris⋅Cl, pH 8.0, ice cold1 mg/ml lysozyme (Roche Molecular Systems), freshly dissolved 10 mg in 10 ml

of 250 mM Tris⋅Cl, pH 8.0, and filtered through 0.22-µm filter250 mM EDTA, pH 8.0, ice cold50 mM Tris⋅Cl, pH 8.0, ice cold


9.5.18



10 mg/ml DNase I (Roche Molecular Systems; store at −20°C)DMEMPEG 1500 in DMEM supplemented with DMSO (see recipe)Mouse BALB/c myeloma cell line J558L (ECACC #88032902) or another

myeloma cell line50 mg/ml kanamycin50 mM L-histidinol (see recipe)Polyclonal anti-IgSF-CAM antibodyFluorescein-conjugated secondary antibody

25-ml cell culture flasks12-ml and 50-ml polypropylene tubes15- and 50-ml conical polypropylene centrifuge tubes37°C bacterial shaker500-ml Erlenmeyer flaskRefrigerated tabletop centrifuge37°C water bathGlass microscope slidesMicroscope with 1000× magnificationMultipipet trays24- and 96-well tissue culture platesMultipipettor and tipsPlastic wrap (e.g., Saran)96-well plates with V-shaped wells

Additional reagents and equipment for indirect immunofluorescence (UNIT 4.3) andfreezing cells (UNIT 1.1)



NOTE: The following protocol is written for one sample. It is not recommended to handlemore than four samples in parallel.

Day 1: grow transformed bacterial strain1. Streak a glycerol stock of an E. coli strain 803 clone containing the mammalian

expression vector onto an LB agar/ampicillin plate. Grow overnight at 37°C.

E. coli strain 803 (also termed 1106) might be more efficient than other E. coli strains inproducing stable protoplasts (Rassoulzadegan et al., 1982).

The antibiotic has to be adapted to the prokaryotic selection marker of the mammalianexpression vector.

2. Grow myeloma cells in DMEM supplemented with 10% (v/v) FCS in 25-ml cellculture flasks. Aim at a high cell density of ∼1 × 106 cells/ml that is reached on day3 (protoplast fusion requires 5 × 106 cells per sample).

Day 2: grow transformed bacterial cultures3. Inoculate 2 ml LB medium prewarmed at 37°C in a 12-ml polypropylene tube with

a single E. coli colony from the freshly streaked LB agar/ampicillin plate. Add 2 µlof 50 mg/ml ampicillin. Grow for 4 hr at 250 rpm in a 37°C bacterial shaker.


9.5.19

Cell Adhesion

4. Dilute 100 µl of cell culture into a 500-ml Erlenmeyer flask with 100 ml of LBmedium and 100 µl of 50 mg/ml ampicillin. Grow to an OD600 of ∼0.6. Start checkingthe optical density after 3 hr.

5. After OD600 ∼0.6 is reached, add 200 µl of 60 mg/ml chloramphenicol to a finalconcentration of 120 µg/ml. Grow overnight at 250 rpm in a 37°C bacterial shaker.

Plasmids carrying the colE1 origin of replication can be amplified in the presence ofchloramphenicol (Hershfield et al., 1974).

Day 3: harvest bacterial cells and form protoplasts6. Transfer the overnight culture into two 50-ml conical polypropylene centrifuge tubes

and centrifuge 10 min at 2500 × g, 4°C.

7. In the meantime, prewarm 20 ml DMEM supplemented with 10% sucrose and 10mM MgCl2 in a 50-ml polypropylene tube in a 37°C water bath.

8. Pour off the supernatants of the spun culture.

Prepare protoplasts9. From here on proceed in a sterile laminar flow bench. Vortex and combine the two

bacterial pellets in 2.5 ml ice-cold 20% sucrose in 50 mM Tris⋅Cl, pH 8.0.

10. Add 500 µl ice-cold 1 mg/ml lysozyme in 250 mM Tris⋅Cl, pH 8.0, mix gently (swirl),and incubate 5 min on ice.

11. Add 1 ml ice-cold 250 mM EDTA, pH 8.0, gently swirl, and store on ice 5 min.

12. Add 1 ml ice-cold 50 mM Tris⋅Cl, pH 8.0, gently swirl, and incubate 10 min at roomtemperature. During this incubation period, mount 10 µl of the sample on a glassmicroscope slide and analyze protoplast formation under a microscope.

A microscope with 1000× magnification is required to distinguish between sphericalprotoplasts and rod-shaped bacteria. At the end of incubation, ∼90% protoplasts shouldbe formed.

13. Add 20 ml DMEM supplemented with 10% sucrose and 10 mM MgCl2 very slowlyto the protoplast preparation. To do this, swirl protoplasts gently, start adding dropsof DMEM supplemented with 10% sucrose and 10 mM MgCl2, and slowly increasethe added volume.

The prepared protoplasts are fragile and need to be handled with care. Protoplast lysis isindicated by an increasing viscosity of the preparation due to the release of genomic DNA.The preparation can be analyzed using a microscope as above.

14. Add 40 µl of 10 mg/ml DNase I and incubate 15 min at room temperature.

Addition of DNase I reduces the viscosity of the protoplast preparation.

Prepare for fusions15. In the meantime, prewarm the following in separate 50-ml propylene tubes in a 37°C

water bath:

15 ml DMEM10 ml DMEM supplemented with 10% (v/v) FCS50 ml DMEM supplemented with 10% (v/v) FCS.

Thaw at room temperature:

2 ml PEG 1500 in DMEM supplemented with DMSO.


9.5.20



16. Centrifuge protoplast preparation in the 50-ml polypropylene tube 30 min at 2500 ×g, room temperature.

17. In the meantime, transfer 5 × 106 mouse BALB/c myeloma cells into 15-ml conicalpolypropylene centrifuge tubes and centrifuge 10 min at 500 × g, room temperature.Aspirate the supernatant and resuspend the cells in 5 ml prewarmed DMEM.

The myeloma cell preparation should be serum-free.

Other myeloma cell lines that have been transfected by protoplast fusion or electroporationinclude mouse P3-X63Ag8.653, mouse Sp2/0-Ag14, mouse NSO, and rat YB2/0 (Gillies etal., 1989; Nakatani et al., 1989; Bebbington et al., 1992; Shitara et al., 1994).

18. Pour off the supernatant of the centrifuged protoplast pellet.

The protoplast pellet should have a smooth surface.

Carry out fusions19. Slowly layer the myeloma cell preparation on top of the protoplast pellet in the 50-ml

conical polypropylene centrifuge tube. Centrifuge 10 min at 500 × g, room tempera-ture.

20. Aspirate the supernatant. Mix cell and protoplast pellet by hand-flicking the tube andtapping it on the benchtop.

21. Add 2 ml PEG 1500 in DMEM supplemented with DMSO. Resuspend the pellet bypipetting up and down several times.

22. After addition of the PEG solution (∼1 to 2 min), very slowly add 10 ml prewarmedDMEM. To do this, swirl protoplasts gently, start adding drops of DMEM, and slowlyincrease the added volume.

23. Add 10 ml prewarmed DMEM supplemented with 10% FCS, swirl gently, andcentrifuge 10 min at 500 × g, room temperature.

24. Aspirate the supernatant, resuspend the pellet in 50 ml prewarmed DMEM supple-mented with 10% FCS, and add 100 µl of 50 mg/ml kanamycin.

25. Pour into a multipipet tray and distribute among five 96-well tissue culture plates byadding 100 µl/well using a multipipettor. Wrap tissue culture plates in plastic wrapand incubate for 48 hr in a 37°C, 10% CO2 humidified incubator.

Day 5: select transfected cells26. After 48 hr, prepare selection medium by adding 10 ml prewarmed 50 mM L-histid-

inol to 40 ml prewarmed DMEM supplemented with 10% FCS. Add 100 µl of 50mg/ml kanamycin. Pour into a multipipet tray and add 100 µl/well using a multipipet.Rewrap tissue culture plates in plastic wrap and continue incubation in a 37°C, 10%CO2 humidified incubator.

The antibiotic, here L-histidinol, has to be adapted to the eukaryotic selection marker ofthe mammalian expression vector. Only transfected myeloma cells will survive the treat-ment with L-histidinol.

27. Examine plates visually for clones ∼10 days after selection medium is added.

No medium change or any other treatment is necessary during this time

Identify IgSF-CAM expressing clones28. Once a clone becomes clearly visible by eye, analyze it for the expression of

IgSF-CAM by indirect immunofluorescence staining (UNIT 4.3). Transfer ≤50% of thecloned cells to a well of a 96-well plate with V-shaped wells and perform indirect


9.5.21

Cell Adhesion

immunofluorescence analysis with a polyclonal anti-IgSF-CAM and a fluorescein-conjugated secondary antibody. Identify clones that are expressing IgSF-CAM.

29. Expand positive clones into 24-well tissue culture plates.

30. Subclone positive clones by limiting dilution in 96-well tissue culture plates.

31. Maintain positive subclones in selection medium, e.g., 5 mM L-histidinol in DMEMsupplemented with 10% FCS. Store backup cells frozen in liquid nitrogen usingstandard procedures (UNIT 1.1).

Cells from positive subclones are used in myeloma cell aggregation assays.

BASICPROTOCOL 5

Detecting Cis-Interactions between IgSF-CAMs by Chemical Cross-Linking

The following basic protocols describe methods for the detection of cis-interactions, i.e.,interactions between proteins that reside in the same membrane. In the chemical cross-linking methods described in this protocol, bifunctional reagents are used to establishcovalent cross-bridges between associated proteins. The covalently linked protein com-plexes are then analyzed by SDS-PAGE (UNIT 6.1) and immunoblots (UNIT 6.2), usingspecific antibodies. In the antibody-induced co-capping methods (see Basic Protocol 6),a hypothesized cis-interaction between two cell surface proteins is evaluated by inducinga redistribution of one molecule and testing whether the putative binding protein follows.For both methods, the cells analyzed should be cultured at low density, in order to preventcontact between cells. Under these conditions, close associations of proteins are onlypossible between proteins residing in the membrane of the same cell.

Chemical cross-linking joins two molecules by means of a cross-linking reagent. Themethod critically depends on the cultivation of the cells of interest as single cells at lowdensity to avoid the formation of cell-cell contacts. Moreover, the structural integrity ofthe cells should be maintained throughout the procedure. The use of hydrophilic, mem-brane-impermeable bifunctional cross-linking reagents restricts the cross-linking to ex-tracellular domains of membrane proteins. Considering the close spatial associationbetween proteins interacting within the same membrane, a high degree of specificity ofchemical cross-linking is mandatory.

The specificity of chemical cross-linking is mainly determined by the chemical reactivityof the functional groups of the cross-linking reagent and the length of the spacer separatingthe reactive groups. The N-succinimidyl group combines efficient reactivity with a highselectivity for primary amino groups, thereby limiting the cross-linking to lysine sidechains at the surface of proteins engaged in interactions. Since most IgSF-CAMs containmultiple lysine residues in their extracellular domains, the amino-group-specific homo-bifunctional N-succinimide-derivatives are suitable reagents for the detection of cis-com-plexes formed between such molecules. The following protocol will primarily focus onthe application of hydrophilic homobifunctional di-N-succinimidyl derivatives with rela-tively short (0.6 to 1.2 nm) spacer sequences separating the reactive groups. In order toenhance the specificity of the chemical cross-linking reaction to stably associated pro-teins, lateral movement in the cell membrane is reduced by performing the cross-linkingreaction on ice. Ideal spacer length of the cross-linking reagent, optimal concentrationsof cross-linkers, and reaction times strongly depend on the specific system that is analyzedand must be evaluated empirically. The parameters described below turned out to beoptimal in many experimental situations and represent a good starting point for furtheroptimizations. After quenching the cross-linking reaction, cells are lysed and the cross-linked complexes ideally isolated by immunoprecipitation (UNIT 7.2) using specific anti-bodies against the molecule of interest. Immunoprecipitates are separated by SDS-PAGEand cross-linked complexes can be detected by immunoblot analysis. The presence of


9.5.22



known binding partners within the cross-linked complexes can be addressed by immuno-chemical techniques, which require only low amounts of proteins. However, immuno-chemical analysis clearly restricts the detection of cross-linked binding partners to knownmolecules against which antibodies are available. Scaling-up of the procedure may resultin the isolation of sufficient amounts of cross-linked material for subsequent microse-quencing allowing the detection of novel binding partners.



Materials

Cells of interest growing in tissue culture at low densityPBS with Ca2+/Mg2+ (see recipe)100 mM cross-linking reagent (see recipe):Bis(sulfosuccinimidyl)suberate (BS3) in water

Disuccinimidyl tartrate (DST) in water-free DMSO

Disulfo disuccinimidyl tartrate (Sulfo-DST) in water

3, 3′-Dithiobis(sulfosuccinimidyl propionate) (DTSSP) in water5 mM EDTA in Ca2+/Mg2+-free PBS1 M glycine solution in water, pH 8.0Lysis buffer (see recipe)Primary antibody: serum, purified immunoglobulin, or purified immunoglobulin

immobilized on agarose or Sepharose matrixProtein A or protein G coupled to agarose or Sepharose matrix (optional)Wash buffer (see recipe)Sample buffer for SDS-PAGE (APPENDIX 2A)

10-cm tissue culture dishes precoated with poly-D-lysine combined with additionalsubstrates, such as laminin (see Support Protocols 10 and 12)

Horizontal shakerCell scraper2-ml microcentrifuge tubesEnd-over-end rotator (model 750)100- or 200-µl and 500-µl Hamilton syringe and 22-G needle

Additional reagents and equipment for SDS-PAGE (UNIT 6.1), immunoblot analysis(UNIT 6.2), and immunoprecipitation (UNIT 7.2)

Prepare cells1. Prior to the experiment, seed cells onto 10-cm tissue culture dishes at a low density.

Plate slowly dividing (division rate of <1 division in 48 hr) or nondividing cells, e.g.,primary neurons, 12 to 16 hr prior to the experiment at a density of 104 cells per cm2

on cell culture dishes precoated with poly-D-lysine (Support Protocol 10) combinedwith additional substrates, such as laminin (see Support Protocol 12).

Fast dividing cells, especially fast-growing cell lines, are problematic in this experimentas extensive contacts are formed between the progeny of cell divisions. With COS or HEK293 cells, cultures of mainly single cells can be obtained by plating the cells 4 to 6 hr priorto the experiment on poly-D-lysine coated cell culture dishes in a density of 1-2 × 104 cellsper cm2. For the final passage of cells, do not trypsinize cells. Most cell lines can easily bedetached when cultured on untreated cell culture plastic by incubation for a few minutesin 5 mM EDTA in Ca2+/Mg2+-free PBS at room temperature. Care must be taken to generatesingle-cell suspensions without any cell aggregates by careful trituration using a large-bore


9.5.23

Cell Adhesion

pipet tip. Glass pipets (Pasteur pipets) are not recommended, since the sharp glass edgesand the strong shearing forces damage the cells resulting in the formation of clumps andaggregates.

2. Use one 10-cm tissue culture dish (containing ∼106 cells) for each reaction. In thefirst set of experiments, include three different concentrations of cross-linkingreagent and three to four different reaction times, resulting in a total of nine to twelveindividual samples.

3. Prior to the experiment, check cells for homogenous distribution under an invertedmicroscope that allows the examination of living cells. Do not use cultures containingaggregates or clumps of cells.

4. Remove culture medium and wash the cells three times with PBS containingCa2+/Mg2+. Add 10 ml PBS with Ca2+/Mg2+ and incubate 5 to 10 min on ice.

Incubation on ice not only reduces lateral movements of proteins within the cell membranebut also decreases endocytosis of membrane proteins.

The cells are living and therefore, during this and subsequent steps, 5 through 8, are veryfragile, especially primary neurons that have the tendency to detach spontaneously duringthe procedure. All steps must be carried out with the utmost care to protect the culturesfrom mechanical stress.

Cross-link cells5. During the incubation period of step 4, freshly prepare 10 ml each of 0.1 mM, 1 mM,

and 10 mM cross-linker solution per dish in PBS with Ca2+/Mg2+, using the 100 mMDST stock solution in water-free DMSO.

The concentration of the cross-linking reagent is a very critical parameter. Most publishedprotocols use concentrations of ∼1 mM for efficient cross-linking. For a first set ofexperiments, the authors recommend 0.1 mM, 1 mM, and 10 mM for the reagents DST andDSSP.

The cross-linker solution in PBS with Ca2+/Mg2+ is unstable at room temperature andshould be kept on ice for ≤5 to 10 min prior to adding to cells.

6. Remove PBS from cells and add the prechilled cross-linker solution carefully to thecells. Carry out the cross-linking reaction on ice with gentle shaking (20 to 30 rpm)on a horizontal shaker.

Tilt cell culture dish slight and add cross-linker solution at the edge of the dish with a pipet.Distribute the cross-linker solution carefully over the entire surface of the dish.

Quench reaction and lyse cells7. Quench the reaction after 0, 5, 15, and 45 min by adding 0.5 ml of 1 M glycine

solution, pH 8.0 (final glycine concentration is 50 mM). Perform the quenchingreaction for 15 min.

The reaction time is a critical parameter regarding specificity and yields of chemicalcross-linking. Quenching of the reaction in an initial experiment after 0, 5, 15, and 45 minresults in a “kinetic profile” of the cross-linking reaction. This “kinetic profile” is oftenvaluable, not only for the optimization of the reaction time as a parameter but also in theinterpretation of the results.

8. Completely remove the reaction mixture from the cells without detaching them andadd 2 ml of lysis buffer per dish. Remove cells from the culture dish with a cell-scraperand lyse them for 30 min on ice on a horizontal shaker.

9. Transfer cell lysate to 2-ml microcentrifuge tubes and microcentrifuge 15 min at12,000 to 14,000 rpm, 4°C.


9.5.24



This centrifugation step clears the cell lysates from nuclei and cell debris. Still, the clearedlysates frequently have an opaque appearance. The lysates can be frozen and kept forseveral weeks at −20°C.

Immunoprecipitate cross-linked proteins10. In 2-ml microcentrifuge tubes, prepare the primary antibody for immunoprecipita-

tion.

Primary antibodies for immunoprecipitation can be used in the form of whole serum,purified immunoglobulins, or coupled to an agarose or Sepharose matrix (for coupling ofpurified immunoglobulins to cyanogen bromide agarose or Sepharose see Support Protocol 1).

Prior to the addition of the cell lysate, preincubate whole serum with Sepharose-coupledprotein A (ideal for rabbit antisera) or protein G (for goat or sheep antisera) in a ratioserum/protein A/G Sepharose of 2:1 and purified immunoglobulins with protein A/G matrixin a ratio of 5 µg immunoglobulin/µl matrix for 30 min at room temperature. For an efficientimmunoprecipitation ∼10 to 20 µg of specific immunoglobulin are required. A total matrixvolume between 10 and 20 µl is recommended.

11. Add the cleared cell lysate to the primary antibody and rotate for 4 to 16 hrend-over-head at 4°C.

12. Microcentrifuge samples for 1 min at 14,000 rpm, 4°C and carefully remove thesupernatant; keep supernatant and store at −20°C.

13. Resuspend matrix in 1 ml wash buffer, vortex for 20 sec, and microcentrifuge 30 secat 14,000 rpm, 4°C. Repeat the wash step three times and carefully remove thesupernatant after final wash step. Remove the residual fluid from the matrix pelletwith a 100- or 200-µl Hamilton syringe without aspirating matrix material.

14. Elute material bound to the matrix in 50 to 100 µl sample buffer for SDS-PAGE byboiling 5 min. Microcentrifuge samples for 1 min at 14,000 rpm, room temperature.

After boiling, the protein sample in SDS-PAGE sample buffer can be stored several monthsat −20°C or years at −70°C.

15. Analyze protein samples by SDS-PAGE (UNIT 6.1) and detect the cross-linked com-plexes by immunoblot analysis (UNIT 6.2).

BASICPROTOCOL 6

Detecting Cis-Interactions between IgSF-CAMs by Antibody-InducedCo-Capping

The lateral mobility of integral membrane proteins within the plane of the cell membraneallows the induction of large clusters of molecules by cross-linking their extracellularmoieties with antibodies. This phenomenon is termed “antibody-induced capping”.Interactions between two cell surface proteins in the plane of the same membrane(cis-interactions) can be addressed by examining the effects of antibody-induced cappingof one molecule on the distribution of the other molecule. Reciprocal co-capping orco-distribution of two molecules after induction of capping with antibodies against oneof them strongly indicates a (direct or indirect) cis-interaction. The following protocoldescribes a basic method for the induction of capping by antibodies and the detection ofco-capping by fluorescence microscopy.

Cells of interest are cultivated at low density to avoid the formation of extensive cell-cellcontacts. Incubation with the primary antibody against the first molecule (A) is performedon intact, live cells in the cold to reduce lateral movement of membrane proteins.Following the incubation with primary antibody against molecule A, unbound antibodyis removed. Bound primary antibody is detected by a fluorochrome-conjugated secondary


9.5.25

Cell Adhesion

antibody against the animal species in which the anti-A primary antibody was raised.Subsequently raising the temperature to 37°C restores the lateral mobility of proteins inthe cell membrane and results in the formation of clusters of molecule A due toantibody-mediated cross-linking. The cells are washed, fixed, and counter-stained with aprimary antibody against the putative binding partner of molecule A (molecule B). To thisend, the primary antibody against molecule B must be raised in a different species thanthe anti-A antibody. The anti-B antibody is detected by a secondary antibody conjugatedto a different chromophore than the secondary antibody against molecule A. Care shouldbe taken that the two antibody combinations do not cross-react. Further, the emissionwavelengths of the chromophores coupled to the secondary antibodies must be distinctto allow a complete separation of the signals by the use of narrow band-pass filters in thesubsequent examination by immunofluorescence microscopy. Once the conditions forantibody-induced capping and for observing specific immunolocalization have beenestablished, co-localization of the fluorescence signals elicited by the secondary antibod-ies allows the detection of co-clustering of molecules A and B on the cell surface.Quantitative analysis of the localization of the fluorescent signals requires a spatialresolution not normally achieved by standard light microscopes. To this end, due to itssuperior resolution power, confocal laser scanning microscopy represents a powerfultechnique for the quantitative assessment of co-distribution induced by antibody-inducedcapping.

Materials

Cells of interest growing in tissue culture (e.g., nondividing cells or primaryneurons)

Cell culture medium (used for the cells of interest) without serumPBS (see recipe)Hank’s Balanced Salt Solution (HBSS; APPENDIX 2A)HBSS/1% FCS: HBSS containing 1% (v/v) fetal calf serum (FCS)Primary antibodies (serum or purified immunoglobulin) for molecules A and BSecondary antibodies of the appropriate sources coupled to different fluorescent

dyes4× Fixative solution (see recipe)Vectashield mounting medium for fluorescence (H-1000, Vector Laboratories)10-cm tissue culture dishes12-mm no. 1 round glass coverslips, sterilized by autoclaving or soaking in 70%

ethanol for 1 hr and precoated with poly-D-lysine (see Support Protocol 10)Watchmaker’s forceps24-well tissue culture platesMicroscope slidesNail polishFluorescence microscope with 63× and/or 100× oil immersion objectives



Prepare cells1. One day prior to the experiment, seed cells at low density onto 10-cm cell culture

dishes, each containing 15 to 20 sterilized poly-D-lysine coated 12-mm round glasscoverslips. Plate nondividing cells or primary neurons 12 to 16 hr prior to the


9.5.26



experiment at a density of 2 × 104 cells per cm2 and fast-growing cell-lines 8 to 12 hrprior to the experiment at a density of 104 cells per cm2.

These cell densities should result in ∼10% confluency at the time of the experiment.

Coating of glass coverslips with poly-D-lysine is always recommended, in order to preventcell detachment during the procedure.

2. Two to 4 hr prior to the experiment, transfer the glass coverslips with watchmaker’sforceps from the 10-cm cell culture dish to wells 24-well tissue culture platescontaining 1 ml cell culture medium per well. Incubate at 37°C

Aseptic technique is recommended, but not mandatory.

3. After 2 to 4 hr of incubation, remove the culture medium and wash the cells threetimes with 1 ml HBSS at room temperature, each time by adding 1 ml of HBSS,incubating 1 min, and aspirating wash solution. Add 200 µl HBSS/1% FCS per welland incubate the 24-well plate for 10 min on ice.

Do not let the cells dry throughout the procedure. The incubation with HBSS/1% FCS onice will block potential unspecific sites for antibody adsorption. Incubation on ice not onlyreduces lateral movements of proteins within the cell membrane but also decreasesendocytosis of membrane proteins.

The cells are living and therefore, during this and subsequent steps are very fragile,especially primary neurons that have the tendency to detach spontaneously during theprocedure. Carry out all steps with the utmost care to protect the cultures from mechanicalstress.

Prepare and bind primary antibody for molecule A4. Prepare the appropriate dilutions of primary antibody and control antibody to

molecule A in HBSS/1% FCS in microcentrifuge tubes and centrifuge for 1 min atmaximum speed. Transfer supernatants to new tubes and put primary antibodysolutions on ice ≥5 min prior to use.

For co-capping experiments the primary antibodies are generally applied at higherconcentrations than for standard immunofluorescence protocols. Titrate optimal antibodyconcentrations for each experimental system individually. As a rule, dilute affinity-purifiedantibodies to a range of 5 to 20 µg/ml and antisera between 1:20 and 1:200. If using acommercially available antibody, multiply the concentration recommended for im-munofluorescence staining by a factor of five. For a first set of experiments, try a range ofdilutions and test for potential cross-reactivity of the primary antibodies in advance. Addnegative controls to ensure specificity of antibody-induced capping. To this end, preimmuneserum or purified antibodies from preimmune serum are required.

Antibody dilution is a critical parameter regarding cross-reactivity: this phenomenon mayoccur at the high concentrations that are used for co-capping experiments even if absentat concentrations based on own experience or recommended by manufacturers for standardimmunofluorescence protocols.

5. Remove HBSS/1% FCS from cells and add 200 µl of cold anti-A antibody inHBSS/1% FCS to each well. Incubate with the primary anti-A antibody for 30 minon ice.

Prepare and bind secondary antibody for molecule A6. Dilute fluorochrome-conjugated secondary antibody in HBSS/1% FCS in microcen-

trifuge tubes and centrifuge for 1 min at maximum speed. Transfer supernatants tonew tubes and place secondary antibody solutions on ice ≥5 min prior to use.

Commercially available preparations of secondary antibodies must be tested for cross-re-activity with the primary and secondary antibodies against molecule B in advance. Even


9.5.27

Cell Adhesion

if manufacturers assure that no such cross-reactivity occurs, consider that the concentra-tions of secondary antibodies used in co-capping experiments are well above thosenormally applied for immunofluorescence staining. Typical dilutions of commercial prepa-ration are between 1:20 and 1:200 (∼5 to 10 times higher than for standard immunofluo-rescence applications).

7. Aspirate primary antibody solution and wash cells two times with ice-cold HBSS.Add 250 µl/well of secondary antibody and incubate for 30 min on ice. Protect fromlight.

8. Remove the solution containing the secondary antibody and wash the cells two timeswith ice-cold HBSS. Add 200 µl of ice-cold cell culture medium without serum andput the 24-well cell culture plate for 30 min in a 37°C, 5% CO2 humidified incubator.

Never use HBSS or PBS for this incubation step. The phosphate buffer system present inHBSS and PBS is insufficient to buffer the pH efficiently in a 5% CO2 atmosphere. The pHwould drop too much and lead to false results. Always use the cell culture medium normallyapplied for the cell cultures of interest without serum. When stored on ice, keep the cellculture medium in air-tight tubes.

Fix cells9. Add 66.7 µl of 4× fixative solution to each well and allow cells to fix for 15 µin at

37°C in the dark.

A frequent source of experimental artifacts in co-capping experiments is fixation. It isnecessary to block any further lateral movement of the capped proteins after fixation. Thefixation in culture medium with 2% formaldehyde/0.1% glutaraldehyde results in totalimmobilization of membrane proteins without permeabilization of the cell membrane (fora detailed discussion, see Dubreuil et al., 1996).

10. Aspirate fixative and wash cells two times with PBS. Remove the PBS after thesecond wash step and add 500 µl PBS/1% FCS. Incubate for 15 min at roomtemperature.

Prepare and bind primary antibody for molecule B11. Prepare dilutions of the primary antibody for molecule B in PBS/1% FCS as

described in step 4.

For the counter-staining (here of molecule B), the primary antibodies are generally appliedin concentrations comparable to standard immunofluorescence protocols. Titrate optimalantibody concentrations for the counter-staining in a way that similar intensities offluorescence signals result for both molecules. As a rule, dilute affinity-purified antibodiesin a range of 1 to 10 µg/ml, purified Fab fragments 5 to 20 µg/ml, and antisera between1:100 and 1:1000.

If using a commercially available antibody, the concentration recommended for im-munofluorescence staining is a good starting point for the optimization of the counter-staining. For a first set of experiments, try a range of dilutions and test again for potentialcross-reactivity of the antibodies in advance. Add negative controls to ensure specificity ofthe counter-staining. In addition to preimmune serum or purified antibodies from preim-mune serum, include further primary antibodies against molecules for which no co-cappingwith molecule A is expected in the experiment. The specificity of co-capping is the mostcritical issue in the whole experiment.

12. Remove the PBS/1% FCS and add 200 µl/well of diluted primary anti-B antibody inPBS/1% FCS. Incubate 1 hr at room temperature. Protect from light with aluminumfoil or place 24-well plate in a drawer.


9.5.28



Prepare and bind secondary antibody for molecule B13. Dilute the secondary antibody against anti-B antibody in PBS/1% FCS as described

in step 6.

If commercially available preparations of secondary antibodies are used for the counter-staining, dilutions between 1:100 and 1:500 are recommended in most cases. Theseconcentrations are similar to those used for standard immunofluorescence applications.The issue of cross-reactivity with the primary and secondary antibodies against moleculeB is much less critical than in case of the antibody used for capping but have to be testedanyway to use the system in reverse order to detect reciprocal co-capping (see below).

14. Remove primary anti-B antibody solution and wash cells two times with PBS/1%FCS. Add 200 µl/well secondary anti-B antibody in PBS/1% FCS and incubate 45min at room temperature. Protect from light.

Fix cells15. Wash cells two times with PBS/1% FCS and one time in PBS only. Add 250 µl of 1×

fixative solution (2% formaldehyde/0.1% glutaraldehyde in PBS) and let cells fix for5 min at room temperature in the dark.

16. Remove fixative and wash cells two times with PBS.

The samples can be stored protected from light for up to 1 day at 4°C.

Mount and examine cells17. Label microscope slides and place 1 drop of Vectashield mounting medium onto slide.

Carefully remove each coverslip from the 24-well plate with watchmaker’s forcepsand blot excess fluid by touching edge with a paper towel. Invert coverslip, cell-sidedown onto mounting medium. Do not apply pressure. Blot excess mounting mediumwith a paper towel and allow slides to dry 5 min at room temperature in the dark. Sealaround the rim of the coverslip with nail polish.

18. Examine specimen on a standard fluorescence microscope using a 63× or 100× oilimmersion objective.

Due to its superior resolution power, confocal laser scanning microscopy represents thepreferred technique for the quantitative assessment of capping and co-capping.

BASICPROTOCOL 7

Neurite Outgrowth Assay

This protocol describes a method to quantify the growth of neurites from cultured neurons.Neurite outgrowth assays are used to determine a potential role of an IgSF-CAM as asubstratum for neurite outgrowth. The authors describe here the use of embryonic chickensensory neurons (Stoeckli et al., 1991), however depending on the substratum to be testedand the responsiveness of the cells, other neurons such as tectal, cerebellar, and spinalcord neurons, may be used as well. It is important that for these assays the neurons areplated at a very low density in order to prevent contact between neurons and their axons,as well as contact between neurons and non-neuronal cells. Only then does the measuredneurite length reflect the neurite outgrowth-promoting activity of the substratum used.

Many IgSF-CAMs were found to be potent neurite outgrowth-promoting substrates forvarious types of neurites. This protocol is an example of the one used for low-densitycultures of dorsal root ganglia neurons on an IgSF-CAM substrate. Low-density cultures(Stoeckli et al., 1991, 1996), rather than explants or high-density cultures, are used tominimize effects derived from cell-cell contacts. To achieve reproducible results forgrowth assays use serum-free, chemically defined media wherever possible.


9.5.29

Cell Adhesion

Many commercially available software packages (e.g., NIH Image, Metamorph) provideconvenient methods for neurite length measurements. The goal of this protocol, therefore,is not the explication of a particular method of neurite length measurement, but rather thediscussion of basic requirements and principles that need to be fulfilled to obtainreproducible values.

Materials

Chicken embryos (E8 to E10)0.5% (v/v) glucose in PBS0.25% (w/v) trypsin in PBS without Ca2+/Mg2+ (Life Technologies)Serum-free culture medium (see recipe)

Tissue culture dishes coated with substrate of choice (see Support Protocol 8)Sterile dissecting tools15-ml centrifuge tubesFire-polished Pasteur pipet, ∼0.3-mm diameter boreNeubauer chamber for cell counting (Fig. 1.1.1)35-mm cell culture dishesImage analysis software and required equipmentAdditional reagents and equipment for counting cells (UNIT 1.1)

Dissect dorsal root ganglia and process cells1. Coat 35-mm tissue culture dishes with appropriate substrate according to Support

Protocol 8, 9, 10, 11, or 12.

2. Dissect dorsal root ganglia (DRG) from 10-day-old chicken embryos (Sondereggeret al., 1985) with sterile dissecting tools and collect DRGs in a noncoated 35-mmtissue culture dish on ice in 1 ml of 0.5% glucose in PBS.

Younger embryos can also be used, but the dissection before E8 requires more skills andpractice.

3. Transfer DRG to a 15-ml centrifuge tube and centrifuge 3 to 5 min at 300 to 500 ×g, room temperature.

4. Carefully remove supernatant and add 2 ml of 0.25% trypsin solution to pellet.Resuspend ganglia and incubate 25 min in 37°C water bath.

5. Centrifuge DRG for 3 to 5 min at 300 to 500 × g, room temperature, decant trypsinsolution supernatant, and add 1 ml serum-free culture medium.

6. Use a fire-polished Pasteur pipet with an opening of ∼0.3-mm diameter to mechani-cally dissociate (triturate) the ganglia. Continue until no cell clumps are visible by eye.

Less than ten passages should be sufficient to get a single cell suspension.

Count and plate cells7. Count an aliquot of the cell suspension in a Neubauer chamber (UNIT 1.1).

8. Obtain a cell suspension dilution of 150,000 cells/ml. Plate 1 ml of cell suspensionper 35-mm cell culture dish.

9. Incubate cultures for 24 to 30 hr in a 37°C, 5% CO2 humidified incubator.

Axons reach their maximal length after 24 to 30 hr. However, growth rate and onset of axongrowth are substratum-dependent.

Measure and count neurites10. At the appropriate time, measure neurite length for neurites extending from single

cell bodies without contact to other neurites (see Fig.9.5.1 and legend).


9.5.30

A prerequisite for reliable neurite length measurements are low-density cultures of suffi-cient and reproducible quality. Care has to be taken to avoid the measurement of neuritefascicles rather than single neurites, as this confuses the results.

Fasciculation as well as cell-cell contact positively influences neurite length. Thus, foraccurate determination of the neurite outgrowth-promoting capacity of a particularsubstrate or experimental condition, only single neurites extending from single cell bodieswithout contact to other neurites and especially without contact to non-neuronal cellsshould be included in the experiment. Cell numbers and culture conditions have to bechosen accordingly.

Figure 9.5.1 In order to be a valid assessment of the neurite outgrowth-promoting qualities of a testsubstrate, the evaluation of neurite outgrowth has to be carried out according to strict standards. Cultureconditions have to be reproducible and identical. Therefore, chemically defined media should be used ratherthan serum containing media. Even minor changes in the composition of media can dramatically altergrowth characteristics and the morphology of neurons (e.g., Savoca et al., 1995). The main criteria in theevaluation of neurite growth-promoting qualities of a test substratum is neurite length. However, keep inmind that only neurite lengths of single axons and not bundles of axons should be measured, as the latterwould reflect a combination of neurite outgrowth promoted by the substratum and neurite growth alongaxons. Neurite length can be assessed either as length of the longest neurite (C) or as total neurite lengthof a neuron, in which case the lengths of all neurites of a neuron are added up and represented as onevalue. Different means can be used to measure the length of a neurite. The most convenient way is the useof a computerized system where one can trace the neurite with the joy stick and the computer willautomatically determine its length. Make sure that the software allows the inclusion of the length of the sidebranches, as they have to be included into the measurements. The comparison between the neurons shownin (A) and (B) clearly demonstrates that the branching of a neurite is an important trait characteristic for agiven substratum. The clear morphological differences between the neurons shown in (A) and (B) arereflected in the length plots (see Fig. 9.5.2). While the curves for the length of the longest neurite and thetotal length of all neurites of a neuron would greatly differ for neurons such as the one shown in (A), theywould be superimposed for neurons like the one shown in (B). In other words, a strong deviation of the twocurves indicates that neurons have, on average, multiple neurites, whereas a small deviation indicates thatthe majority of the neurons have only one neurite. Another way to quantify the morphological differencesbetween neurons is the counting of branch points per neurite as shown in (D). While the neuron shown in(A) has 0 to 2 branch points per neurite, the neuron shown in (B) has 8 branch points (D).


9.5.31

Cell Adhesion

11. Count the number of neurites per neuron, number of branches, and branching order.

Very often the morphology of neurites is substratum-specific. Therefore, the total neuritelength per neuron may only give you a partial account of the differences between distinctsubstrates. Therefore, additional information should be collected, such as number ofneurites per neuron, number of branches per neurite, and branching order (i.e., appearanceof secondary or even tertiary branches).

12. Analyze the growth cone. Measure the area, number of filopodia, and length offilopodia.

Sometimes the analysis of the growth cone morphology is crucial. Characteristics, such asgrowth cone area, number of filopodia, or length of filopodia, can change dramaticallydepending on the substrate used. However, the culture conditions must be controlledcarefully, because many of these characteristics can vary depending on the culture medium

100

90

80

70

60

50

40

30

20

10

0

% n

eurit

es lo

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than

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total neurite length

% n

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es lo

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comparison of the neurite length of the longest neurite

0 25 50 75 100 125 150 175 200 225 250 275 300

Neurite length [micrometer]

100

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60

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0

Figure 9.5.2 Graphic representation of neurite length. The neurite outgrowth-promoting activityof different IgSF-CAMs compared to each other or compared to a control protein can be representedin the type of plot that was introduced by S. Chang and colleagues (1987). In these plots the totallength of all neurites extending from a neuron can be shown. The two curves in (A) represent agood (solid line) versus a poor substrate (dashed line). The same type of plot can also be used torepresent a difference in neurite morphology (B). When the two curves representing the length ofthe longest neurite of a cell (dashed line with squares) and the total length of all neurites of a givencell (solid line with dots) differ a lot from each other, the neuron has most likely several long neurites,whereas cells with only one long neurite have plots where the two curves are almost the same.


9.5.32



as well as on the substratum used. Also, carefully control the origin of the neurons: onlyneurons from the same embryonic age can be compared. Neurites taken from adult animalscan show completely different growth characteristics compared to embryonic neurons. Thisis especially true with respect to the dependence of neurons for trophic factors. Ideally,serum-free, chemically defined media are used in comparative studies of neurite length andmorphology, as the content and concentration of serum components is subject to consid-erable batch-to-batch variability.

13. Represent the quantitative results.

A very common graphic representation of neurite length is the plot introduced by Changet al. (1987). In these graphs the percentage of neurons with a total neurite length longerthan x is easily visible (Fig. 9.5.2). Similarly, the length of the longest neurite per cell orthe number of branches can be represented. The comparison between the curves for totalneurite length per neuron and the length of the longest neurite can be used as a graphicalrepresentation of the difference between neurons with predominantly one neurite that isvery long and neurons with many neurites per neuron that amount to the same total lengthwhen added together (Fig. 9.5.2B).

BASICPROTOCOL 8

Inhibiting CAM-CAM Interactions In VitroOne way to investigate the function of a particular protein is the analysis of changesresulting from blockage of function. This can be done with several methods both in vivoand in vitro. Whereas in vivo experiments are far more difficult and complex to perform,they more likely reflect the true function of the particular CAM because all parametersare the same, all binding partners are present, all interactions are possible, and even themost difficult parameter to test experimentally, the factor time, can be taken into account.However, there are also disadvantages; in vivo assays are far more complicated andtime-consuming than in vitro studies. Most in vivo studies in vertebrates are done in“knock-out” mice, whereby homologous recombination in embryonic stem cells, a geneof interest is replaced by a construct that selects for the cells that have lost the expressionof this gene. These “knock-out” mice can be analyzed for phenotypes resulting from thelack of expression of the gene of interest. While this is certainly a very potent method toanalyze the function of CAMs, there are several major drawbacks; it is time-consuming,expensive, labor-intensive, and requires appropriate facilities with specially trainedexperimenters.

Furthermore, as the analysis of several knock-out lines has shown in the past, somemutations result in early embryonic lethality, in which case the organ of particular interestmay not have developed by the time of death. This is especially true, when the functionof a particular gene is to be analyzed in the nervous system. Alternatively, the lack of onegene may be compensated for, in which case, phenotypes are not detectable or only minorchanges occur that are detectable only by very detailed analyses that often exceed thepotential of one lab.

An alternative to these genetic loss-of-function analyses is an in vivo study where theblockage of function is done at the protein level (e.g., Stoeckli and Landmesser, 1995;Perrin and Stoeckli, 2000). Due to the relatively easy accessibility of the chicken embryo,it is the model of choice for this type of study. Obviously in contrast to the in vivoexperiments described above, this is only possible for functional studies during embryonicdevelopment. Although less expensive and less dependent on an appropriate animalfacility, in vivo studies using chicken embryos as a model system are time-consumingand require a considerable level of technical skills and training.

A description of in vivo loss-of-function studies would exceed the scope of this unit. Forthat reason, only in vitro methods for inhibition of IgSF-CAM function will be discussed.


9.5.33

Cell Adhesion

The blockage of IgSF-CAM function in cultures of dissociated cells or tissue explants ismuch less complex than the inhibition of IgSF-CAM function in vivo; however, the resultsobtained in vitro may not reflect or only partially reflect the function of a particularIgSF-CAM in vivo. The advantages of in vitro experiments are: they are easier to perform,less time-consuming, and more suitable to study a particular question, due to lower degreeof complexity. Assays as the one described in this protocol were used to study the role ofNgCAM (Chang et al., 1987) and axonin-1 (Stoeckli et al., 1991) in the fasciculation ofdorsal root ganglia axons.

Explants of intact dorsal root ganglia (DRG) are grown on a collagen substrate. Underthese culture conditions axons extend from the explants in a radial fashion in the form offascicles. The role of NgCAM as a candidate molecule mediating the axon/axon interac-tion under these conditions has been tested by the addition of Fab from anti-NgCAMantibodies to the culture medium.

Materials

10-day-old chicken embryos (E10)0.5% (w/v) glucose in PBSChemically defined, serum-free cell culture medium (see recipe)Control FabFab against CAM of interest

15-ml centrifuge tubes8-well-slide cell culture dishes (e.g., LabTek, Life Technologies) coated with

IgSF-CAM (see Support Protocol 8)Pasteur pipet or automatic pipettor with 200-µl tips

1. Dissect DRG from E10 embryos, collect in 0.5% glucose in PBS on ice.

2. Transfer DRG to a 15-ml centrifuge tube, and centrifuge for 3 to 5 min at 300-500 ×g, 4°C or room temperature.

3. Decant supernatant and resuspend pellet in 1 ml serum-free cell culture medium bytapping the vial on the bench and swirling the tube.

4. Prepare 8-well-slide cell culture dishes coated with IgSF-CAM according to SupportProtocol 8 by filling the wells with 300 µl medium containing no Fab, control Fab,or Fab against CAM of interest in different concentrations.

Antibody concentrations ranging from 100 to 500 µg/ml for polyclonal and 10 to 500 µg/mlfor monoclonal IgGs have been found useful for in vitro studies.

5. Plate DRG in the smallest possible volume either by using a Pasteur pipet or anautomatic pipettor with a 200-µl tip.

The DRG should be carefully placed on the surface of the dish rather than just put in themedium to facilitate attachment to the substratum. Take care not to scratch the coatedsurface of the cell culture dish.

Carefully avoid shear forces that can destroy the ganglia. For inexperienced investigators,it is best to use an automatic pipettor set at 20 µl.

6. Grow DRG for 40 hr in a 37°C, 5% CO2 humidified incubator.

7. Fix DRG as described in Support Protocol 13 by adding fixative directly to the culturemedium to avoid detachment of the ganglia.


9.5.34



COATING OF CULTURE DISHES

To provide a suitable substratum for neurite growth, tissue culture dishes are coated withcomponents of the extracellular matrix, such as laminin (see Support Protocol 12) orcollagen (see Support Protocol 11). Alternatively, IgSF-CAMs can be used as substratumby either coating them directly onto tissue culture plastic (see Support Protocl 8) or ontop of a nitrocellulose layer (see Support Protocol 9). The application of a nitrocelluloselayer prior to coating of the protein substrate (Lagenaur and Lemmon, 1987; see SupportProtocol 9) is useful to increase the coating efficiency. When glass has to be used insteadof tissue culture plastic, precoating with nitrocellulose can be necessary. For coating withECM components such as collagen (see Support Protocol 11) or laminin (see SupportProtocol 12), glass is an acceptable surface even without precoating.

NOTE: All solutions and equipment must be sterile, and proper sterile technique shouldbe used accordingly. Use filtration to sterilize solutions.

SUPPORTPROTOCOL 8

Coating with IgSF-CAM

Purified IgSF-CAMs can be used to coat culture surfaces for neurite growth assays. Aspurified IgSF-CAMs are usually available in limited amounts, draw a ∼0.5-cm2 circulararea near the center of a 35-mm dish with a lab marker on the bottom outside surface ofthe dish. Then apply the protein to be tested to the marked area of the dish only. Coat therest of the dish with bovine albumin (Albumax). This configuration provides better opticalaccess to the cells than a 24 well plate. It also allows comparison of the growth-promotingeffect of the protein of interest with that of bovine serum albumin in the same dish.

Materials

Protein to be coatedPBS (see recipe)10 mg/ml bovine serum albumin (e.g., Albumax, Life Technologies) in PBS

1. Dissolve the proteins in PBS at concentrations of 10 to 100 µg/ml.

Dilute protein solutions should not be stored for extended periods of time, no more than afew days at 4°C and never frozen. If freezing is required, add 100 µg/ml BSA as a carrierprotein. Check for possible interference with the assay.

2. Pipet 20 µl protein solution per 0.5-cm2 area of the culture dish and spread overmarked area or use sufficient solution to cover entire surface.

If glass is used, the volume per area should be doubled to reach sufficient dissipation ofliquid.

3. Incubate for 2 hr in a 37°C humidified incubator.

4. Aspirate protein solution and rinse the entire surface of the dishes three times withPBS.

5. To saturate the protein adsorbance capacity of the tissue culture plastic or glasscoverslip, incubate dishes for 30 min with a 10 mg/ml BSA solution. Cover the entiresurface of the dish (1 ml per 35-mm culture dish).

The use of BSA purified as fraction V from bovine serum is not advised, as most batchescontain contaminations of endotoxins that are detrimental for cell survival, especially forneurons. The bovine serum albumin sold as Albumax from Life Technologies has given verysatisfactory results.

6. Rinse dishes three times with PBS. Remove PBS immediately before plating cells.For reproducible results, do not allow the coated proteins to dry.

Dishes should be coated immediately before use; storage is not recommended.


9.5.35

Cell Adhesion

SUPPORTPROTOCOL 9

Pre-Coating Glass Surfaces with Nitrocellulose

IgSF-CAMs do not adhere well to glass surfaces unless those surfaces have previouslybeen coated with nitrocellulose.

Materials

MethanolNitrocellulose (e.g., BA-83, Schleicher and Schuell), 0.2-µm pore sizeCoverslips, 22-mm diameter (preclean glass with acetone before coating, dry, and

autoclave)

1. Dissolve a 5-cm2 piece of 0.2-µm pore size nitrocellulose membrane in 17 mlmethanol.

Make sure membrane is completely dissolved. The membrane becomes translucent and isdifficult to see. The solution has to be prepared immediately before use. Do not store.

2. Dilute 300 µl of nitrocellulose membrane/methanol solution with 3.7 ml sterile waterto obtain a coating solution.

3. Coat 22-mm glass coverslips by incubating with 200 µl coating solution for 2 hr ina laminar flow hood. Remove excess coating solution and let coverslips dry beforeuse. Do not store. Proceed with coating with IgSF-CAM (Support Protocol 8).

The same procedure has been used to precoat Thermanox coverslips for electron micro-scopic use.

Tissue culture plastic can be coated with undiluted nitrocellulose solution, using 450 µl ofthe stock solution per 35-mm culture dish.

SUPPORTPROTOCOL 10

Pre-Coating with Poly-D-Lysine

The neurite outgrowth-promoting capacity of laminin is enhanced by precoating culturesurfaces with poly-D-lysine. Alternatively, poly-D-lysine coating alone provides a goodsubstrate for attachment of neurons and nonneuronal cells (also see Basic Protocol 5).

Materials

0.5 mg/ml poly-D-lysine (see recipe)

1. Make a stock solution of 0.5 mg/ml poly-D-lysine in sterile 150 mM sodium borate,pH 8.4. For coating tissue culture plastic, prepare a working solution immediatelyprior to use.

Stock solution may be stored for a few weeks at 4°C. The sodium borate buffer can beautoclaved or filtered before poly-D-lysine is added.

Depending on the type of culture, the coating concentrations range between 10 and 500µg/ml. Dilute the appropriate volume of the poly-D-lysine stock solution with sodium borate.

2. Incubate dishes with 1 ml coating solution per 35-mm cell culture dish overnight ina 37°C humidified incubator.

3. Aspirate coating solution and rinse dishes at least three times with sterile water.Incubate the dishes overnight with sterile water in a 37°C humidified incubator.

The dishes can be used immediately or dried and stored for several weeks.


9.5.36



SUPPORTPROTOCOL 11

Coating with Collagen

Collagen is a good substrate for many non-neuronal cells, but can also be used for axongrowth assays e.g., for motor neurons. For most neuronal populations, laminin is the betterneurite outgrowth promoting substrate than collagen.

Materials

2 mg/ml collagen in 0.1% (v/v) acetic acid, sterile35-mm cell culture dishes

1. Dilute a 2 mg/ml stock solution of collagen in 0.1% acetic acid to get a finalconcentration of 0.25 to 0.5 mg/ml.

2. Coat 35-mm cell culture dishes with 750 µl of 0.25 to 0.5 mg/ml collagen solution.Evaporate the solution by incubating the dishes at 60°C overnight or until dry.

The dishes can be kept for a few weeks in a dry place at room temperature.

SUPPORTPROTOCOL 12

Coating with Laminin

Laminin is a very potent substrate for most neuronal and non-neuronal cells. It can becoated directly to tissue culture plastic and glass surfaces. However, laminin reveals bestneurite outgrowth-promoting capacities when coated on dishes precoated with poly-D-ly-sine (see Support Protocol 10).

Laminin should not be stored in diluted solutions. For best results, keep small aliquots at−20°C, thaw slowly at 4°C, and prepare coating solution immediately before use. Use750 to 1000 µl of 10 to 20 µg/ml solutions of stock per 35-mm cell culture dish. Coatingis done as described in Support Protocol 8 for IgSF-CAMs. Dishes coated with laminincan be stored.

SUPPORTPROTOCOL 13

FIXATION OF CELLS FOR IMMUNOHISTOCHEMICAL STAININGPROCEDURES USING FLUORESCENT ANTIBODIES

In order to avoid the detachment of axons or the collapse of growth cones, the fixativeshould be added directly to the culture medium. For staining procedures involvingfluorescent antibodies, glutaraldehyde can only be used in a very limited concentration,as it is autofluorescent. A concentration of 0.1% (v/v) is compatible with the use offluorescent secondary antibodies. However, some antigens do not tolerate fixation byglutaraldehyde.

Materials

Paraformaldehyde solution (see recipe)Cell culture medium

Add 350 µl of concentrated paraformaldehyde solution to 1 ml cell culture medium toget a final concentration of 2% paraformaldehyde and 0.1% glutaraldehyde. Fix cells for30 min to 1 hr at 37°C or 2 hr to overnight at 4°C.


9.5.37

Cell Adhesion

SUPPORTPROTOCOL 14

FIXATION FOR MORPHOLOGICAL ANALYSIS, NEURITE LENGTHMEASUREMENTS, AND FOR IMMUNOHISTOCHEMISTRY WITHNON-FLUORESCENT SECONDARY ANTIBODIES

If the autofluorescence of glutaraldehyde does not matter, higher concentrations than0.1% (v/v) can be used. Glutaraldehyde has a higher crosslinking activity than formalde-hyde and therefore, is the fixative of choice for good preservation of morphology. Finalconcentrations of up to 1% can be used. Add 1⁄3 vol 4× fixative solution (see recipe) toculture medium. Fix cells 30 min to 1 hr at 37°C or 2 hr to overnight at 4°C. Fornon-neuronal cells that are more strongly attached to the culture dish than neurons withaxons, aspirate the culture medium and add 1× fixative solution.

NOTE: The preservation of antigens may restrict the use of high concentrations ofglutaraldehyde, even if not used in combination with fluorescent secondary antibodies.

REAGENTS AND SOLUTIONS

Use deionized or distilled water in all recipes and protocol steps. For common stock solutions, seeAPPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

Blocking solution (for amino bead coupling)0.5 g bovine serum albumin (Fraction V; Sigma)0.02% (w/v) NaN3

50 mM Tris, pH 8.0, to 100 mlSterilize by filtering through a 0.45-µm filterStore up to 1 month at 4°C

BSA solution, 0.5% (w/v)0.5 g bovine serum albumin (Fraction V; Sigma)0.02% (w/v) NaN3

PBS (see recipe) to 100 mlSterilize by filtering through a 0.45-µm filterStore up to 1 month at 4°C

Ca2+/Mg2+-free buffer (CMF buffer)137 mM NaCl4 mM KCl0.4 mM Na2HPO4⋅2 H2O0.18 mM KH2PO4

12 mM NaHCO3

11 mM glucose, pH 7.2Filter sterilize through 0.22-µm filterStore <2 weeks at 4°C

CaCl2 solution250 mM CaCl2

Dissolve in ultrapure waterSterilize by filtering through a 0.22-µm filterStore at room temperature


9.5.38



Cell culture medium for HEK 293 cellsDMEM containing:2 mM glutamine2.4 g/liter sodium bicarbonate10 mM HEPES, pH 7.510% (v/v) FCSFilter sterilize through 0.45-µm filterStore no more than a few daysWarm to 37°C before adding to cells

Chemically defined, serum-free mediumMEM containing5 mg/ml lipid-rich bovine serum albumin with 0.1% IgG (e.g., AlbuMAX I, Life

Technologies)2 mM L-alynyl-L-glutamine or dipeptide glycyl-L-glutamine (e.g., GlutaMAX I,

Life Technologies)100 µg/ml transferrin10 µg/ml insulin20 ng/ml triiodothyronine40 nM progesterone200 ng/ml corticosterone200 µM putrescine60 nM sodium selenite20 ng/ml nerve growth factor (NGF)Prepare fresh

4-chloro-1-naphthalene solutionStock solution: Dissolve 4-chloro-1-naphthalene in ethanol to give a 3 mg/mlsolution. Store several weeks at −20°C.Working solution: Immediately before use, dilute stock solution in TBS (APPENDIX

2A) and add 30% H2O2 to give final concentrations of 0.5 mg/ml chloronaphthaleneand 0.01% H2O2 in TBS.

Cross-linking reagents100 mM stock solution of cross-linker in water (BS3, Sulfo-DST, and DTSSP) or

water-free DMSO (DST). Stocks in DMSO can be stored at −20°C under drynitrogen or argon for 1 to 2 months.

Bis(sulfosuccinimidyl)suberate (BS3, Pierce)Disuccinimidyl tartrate (DST, Pierce)Disulfo disuccinimidyl tartrate (Sulfo-DST, Pierce)3, 3′-Dithiobis(sulfosuccinimidyl propionate) (DTSSP, Pierce)

These reagents can be stored under water-free nitrogen or argon for up to 1 year at−20°C.

Working solution: Dilute stock solution of cross-linker in PBS with 0.9 mM Ca2+

and 0.5 mM Mg2+ to a concentration of 1 mM. Prepare fresh and do not keep for >1hr on ice.

Fixation solution, 4×40 ml concentrated paraformaldehyde solution (see recipe)5 ml 20× PBS0.4 ml 50% (v/v) glutaraldehyde (reagent grade) in waterH2O to 50 ml

For best results, prepare fresh. If necessary, the fixative can be stored 2 to 3 days at 4°C.


9.5.39

Cell Adhesion

HBS solution50 mM HEPES1.5 mM Na2HPO4

140 mM NaClDissolve in ultrapure waterAdjust pH to 7.05 with 1 M NaOH or 1 M HClSterilize by filtering through a 0.22-µm filterStore for months at 4°C

L-histidinol, 50 mMDissolve 535 mg L-histidinol dihydrochloride (Sigma) in 50 ml of 100 mM HEPES,pH 7.4, pass through a 0.22-µm filter, and store in aliquots for up to 1 year at −20°C.

LB mediumDissolve 10 g NaCl, 10 g bacto-tryptone (Difco), and 5 g yeast extract (Difco) in 1liter water, autoclave, and store at 4°C.

LB/ampicillin agar platesDissolve 15 g bacto-agar in 1 liter LB medium (see recipe) by autoclaving, cooldown to 50°C, add 1 ml of 50 mg/ml ampicillin, mix, and pour into 10-cmpolystyrene plates. Let sit at room temperature overnight and store protected fromlight at 4°C.

Lysis buffer1% (w/v) CHAPS0.1% (w/v) SDS50 mM Tris⋅Cl, pH 7.6 (APPENDIX 2A)150 mM NaCl5 mM EDTA10 µg/ml leupeptin10 µg/ml aprotinin10 µg/ml pepstatin A1 mM PMSF

Protease inhibitors except PMSF are stable in lysis buffer at −20°C for several months.However, PMSF has to be added fresh. Prepare a 100 mM PMSF stock solution inisopropanol and <5 min before use add to lysis buffer to a final concentration of 1 mM.PMSF stock solution can be stored several weeks at −20°C.

Mounting medium10 ml 1M Tris⋅Cl, pH 9.0 (APPENDIX 2A)70 ml glycerol (70% v/v final)5.09 g n-propyl-gallate (0.24 M final)H2O to 100 mlAdjust pH to 9.0Make 1-ml aliquots and store at −20°C

Paraformaldehyde solution, 10% (w/v)Add 5 g paraformaldehyde to 45 ml water, add 75 µl of 1 M NaOH and dissolve byheating to 60°C in a water bath. Let solution cool to room temperature, add waterto a final volume of 50 ml, check that pH does not exceed 7.4. For best results preparefixative immediately before use. Do not store >2 to 3 days at 4°C.


9.5.40



PBS8.00 g NaCl (137 mM final)0.20 g KCl (2.7 mM final)2.16 g Na2HPO4 × 7 H2O (8 mM final)0.20 g KH2PO4 (1.5mM final)990 ml H2OAdjust pH to 7.4 with 1 N HCl or 1 N NaOHAdjust volume to 1 liter with waterFilter sterilize through a 22-mm filterStore at 4°C

PBS with Ca2+/Mg2+

8.00 g NaCl (137 mM)0.20 g KCl (2.7 mM)2.16 g Na2HPO4 × 7 H2O (8 mM)0.20 g KH2PO4 (1.5 mM)0.10 g CaCl2 (0.9 mM)0.10 g MgCl2 × 6 H2O (0.5 mM)990 ml H2OAdjust pH to 7.4 with 1 N HCl or 1 N NaOHAdjust volume to 1 liter with waterFilter sterilize through a 0.22-mm filterStore at 4°C

PEG 1500 in DMEM supplemented with DMSOMelt 42.5 g polyethylene glycol 1500 (PEG 1500) in a microwave oven, add 50 mlDMEM and 10 ml DMSO, pass through a 0.22-µm filter, and store in aliquots upto 1 year at −20°C.

Poly-D-lysine solutionDissolve 0.5 mg/ml poly-D-lysine (Sigma) in sterile 150 mM sodium borate, pH 8.4.Store a few weeks at 4°C.

Wash buffer0.1% (w/v) CHAPS0.1% (w/v) SDS50 mM Tris⋅Cl, pH 7.6 (APPENDIX 2A)150 mM NaCl5 mM EDTA1 mM PMSFAdd <5 min before use

COMMENTARY

Background InformationIgSF-CAMs have been characterized as cru-

cial molecules for both the development andthe normal function of multicellular organisms.They exert their function by mediating the in-teractions between cells under various circum-stances. Interactions mediated by IgSF-CAMsmay determine whether a cell remains where itis or whether it moves somewhere else; theymay be involved in determining the path of cellmigration and the time and the location of the

end of the migratory path. Other IgSF-CAMshave been implicated in processes such as theextravasation of white blood cells, inflamma-tion, wound healing, as well as tumor invasionand metastasis. In the developing nervous sys-tem, IgSF-CAMs play important roles in cellu-lar migration, in the regulation of axonal out-growth and pathfinding, and in synaptogenesis.In the adult nervous system, IgSF-CAMs playa role in the regulation of neural plasticity andin nerve regeneration after injury. Defective


9.5.41

Cell Adhesion

genes of IgSF-CAMs have been found to resultin severe cerebral malformations and mentalretardation.

Recent advances in the characterization ofIgSF-CAMs have revealed that in several casesIgSF-CAMs not only mediate cell-cell adhe-sion by binding to each other, but that they areoften capable of eliciting intracellular signalsupon binding an extracellular ligand. Intracel-lular signals elicited by extracellular ligandcontact of IgSF-CAMs comprise many signalpathways, including the production of secondmessengers and the activation of transmem-brane or intracellular kinases and phosphatases.

The enormous complexity of binding part-ners is the most striking difference between thecalcium-dependent cell adhesion moleculesand the calcium-independent cell adhesionmolecules of the IgSF. Many IgSF-CAMs havebeen found not only to bind to their own kindin so-called homophilic interactions, but alsoto engage in several heterophilic interactions.Therefore, the determination of ligands plays avery important role in the characterization ofIgSF-CAMs. Only by identifying the macro-molecular binding partners of IgSF-CAMs cantheir functional roles in living organisms beelucidated. Some of these very basic studiesdemonstrating interactions between IgSF-CAMs and other binding partners may be ap-plicable to the study of cell surface moleculesin general, including molecules specifically ex-pressed in the immune system, virus receptors,tumor growth markers, or growth factor recep-tors. However, the analyses of the specific func-tional roles of these proteins is beyond thescope of this unit. For these, the reader is re-ferred to more specialized applications.

Trans-interactionsIgSF-CAMs are composed of at least one Ig

fold, which is also the molecular building blockof antibodies (Rader and Sonderegger, 1998).It is therefore conceivable to express IgSF-CAMs in myeloma cells that are specialized inthe production of antibodies. An expressionsystem based on myeloma cells was establishedfor the production of soluble lymphocyte-de-rived cell-surface receptor proteins byTraunecker et al. (1991). This system has alsobeen used for the expression of IgSF-CAMs onthe surface of myeloma cells (Rader et al.,1993). By studying the IgSF-CAMs in a naturalenvironment, i.e., as membrane-bound mole-cules residing in their proper orientation in abiological membrane, trans-interactions be-tween molecules residing in different mem-

branes can be readily detected through cellaggregation. Nonadherent myeloma cells pro-vide an established natural environment forthese studies. The myeloma cell aggregationassay described here has contributed signifi-cantly to the elucidation of the complex inter-action pattern of two IgSF-CAMs, axonin-1and NgCAM, which were found to trans-inter-act homophilically and cis-interact heterophili-cally (Rader et al., 1993; Buchstaller et al.,1996; Rader and Sonderegger, 1998; Sondereg-ger et al., 1998). In addition, the myeloma cellaggregation assay can be used for the structure-to-function analysis of a trans-interaction.Monoclonal antibodies that have been mappedto particular epitopes on the IgSF-CAMs canbe analyzed for interference with myeloma cellaggregation. Defined mutants of IgSF-CAMs,e.g., engineered domain deletion mutants orpathological point mutations, can be expressedin myeloma cells and assessed for their effecton trans-interactions (Freigang et al., 2000).Furthermore, the influence of soluble mole-cules, e.g., ligands or toxins, on trans-interac-tions of IgSF-CAMs can be studied usingmyeloma cell aggregation. The expression inmyeloma cells has also facilitated the produc-tion of soluble variants of IgSF-CAMs andfragments thereof (Rader et al., 1993, 1996).

Chemical cross-linkingChemical cross-linking is a special form of

chemical modification that results in the cova-lent coupling of molecules by a cross-linkingreagent. In contrast to the simple conjugationof two molecules, cross-linking refers to thecovalent coupling of two molecules that un-dergo an interaction with each other. The inter-acting molecules involved can be proteins, pep-tides, nucleic acids, or carbohydrates. Cross-linkers for proteins are bifunctional moleculescontaining two reactive groups that undergoreactions with side chains of amino acids.Cross-linkers can be generally classified intotwo main families, the homobifunctional cross-linkers, which contain two identical functionalgroups, and the heterobifunctional cross-link-ers, which contain different types of reactivemoieties. Simple homobifunctional cross-link-ers, such as dialkyl halides and bis-imidoesters,were introduced in the 1950’s. Since then >300different cross-linking reagents have been syn-thesized and a large number of these are com-mercially available (Ji, 1983; Staros and An-janeyulu, 1989). Recent developments led tothe design and synthesis of cleavable bifunc-tional compounds that allow the recovery of the


9.5.42



individual compounds present within a cross-linked complex after its isolation.

Using bifunctional reagents for cross-link-ing of macromolecules, such as proteins, twodifferent reaction products are generated result-ing from either intramolecular or intermolecu-lar cross-linking. For the study of protein-pro-tein interactions as described in the protocol,intermolecular cross-linking is of interest. Inthe past, intermolecular cross-linking was in-strumental in the investigation of antigen-anti-body complexes, membrane protein structures,and protein-protein interactions at the quarter-nary structural level, e.g., in receptor-ligand ormultienzyme complexes. The application ofchemical cross-linking to identify membranereceptors for macromolecular ligands has be-come an established technique that can be suc-cessfully applied to the analysis of interactionsbetween IgSF-CAMs (Staros, 1988).

The choice of reagents for cross-linkingmembrane proteins must take into considera-tion the particular physicochemical propertiesof biological membranes. A major problem isrepresented by the unspecific, random-colli-sion dependent cross-linking, due to the lateralmobility of membrane proteins. Chemicalcross-linking of membrane proteins should,therefore, generally be carried out in the coldto reduce lateral movements of proteins. Asecond major factor to be considered is thehydrophobicity of the membrane core. Hydro-phobic and hydrophilic reagents have the po-tential to probe different regions of integralmembrane proteins for interactions with bind-ing partners. To study interactions between ex-tracellular domains of IgSF-CAMs, hydro-philic, membrane-impermeable cross-linkersthat bear hydrophilic or charged groups can beused (Buchstaller et al., 1996; Kunz et al., 1998).The limitation of cross-linking to closely asso-ciated binding partners within complexes ofmembrane proteins requires reagents with shortspacers between their reactive groups. How-ever, cross-linkers with spacer lengths of <0.5nm result in poor yields of intermolecular cou-pling, whereas extensive cross-linking with de-creasing specificity is generally observed withreagents with spacer lengths of >1.1 nm (Mid-daugh et al., 1983). The use of a homologousseries of cross-linking compounds is recom-mended for the evaluation of the system.

For efficient cross-linking between extracel-lular domains of membrane proteins, two func-tional groups that differ in reactivity and chemi-cal selectivity are predominantly used, (1) theN-succinimidyl group and (2) the photoactivat-

able aryl azide group. The N-succinimidylgroup combines efficient reactivity with a highselectivity for primary amino groups, therebylimiting the cross-linking to lysine side chainsand free N-termini of proteins. Disuccinimidylderivatives of variable spacer length and hydro-philicity are currently commercially available.Popular hydrophilic disuccinimidyl reagentsare the disuccinimidyl suberate (DSS) deriva-tive bis(sulfosuccinimidyl)suberate (BS3) witha spacer length of 1.14 nm, disuccinimidyltartrate (DST) and its disulfo-variant disulfodisuccinimidyl tartrate (Sulfo-DST) with aspacer length of 0.64 nm. A water-soluble,membrane-impermeable, thiol-cleavabledisuccinimidyl reagent with a spacer length of1.2 nm is represented by 3, 3′-dithiobis(sulfo-succinimidyl propionate) (DTSSP).

The aryl azide group undergoes a UV light–induced chemical reaction resulting in the gen-eration of a nitrene, a highly reactive, short-lived intermediate containing a nitrogen elec-tron sextet (aza-analogon to a carbene).Analogous to carbenes, the nitrenes react im-mediately with their direct environment. Themajor reaction of interest is the insertion intoC-H and N-H bonds resulting in the formationof a new covalent bond. This broad specificityallows the nitrenes to react with virtually allchemical groups present on a protein surface,making this type of cross-linking extremelyefficient. Common nitrene precursors are ary-lazides with absorbance maxima in the long UVregion. Nitro-substitution of the aromatic ringresults in a further shift of absorbance towardslonger wave lengths, away from the absorbancemaxima of proteins. Photosensitive heterobi-functional cross-linkers containing one amine-reactive N-succinimidyl group with the pho-toactivatable aryl azide group are widely usedreagents for the cross-linking of membraneproteins with their receptors (Hermanson,1996). Commercially available products of thisgroup are N-hydroxysulfosuccinimidyl-4-azi-dobenzoate (Sulfo-HSAB, spacer length of 0.9nm) and the highly versatile thiol-cleavablereagent sulfosuccinimidyl 4-(p-azido-phenyldithio) propionate (Sulfo-SADP, with aspacer length of 1.39 nm). For protocols regard-ing the use of these photoreactive cross-linkerssee Jung and Moroi (1983) and Wood andO’Dorisio (1985).

Co-cappingThe detection of cis-interactions by anti-

body-induced co-capping is based on the lateralmobility of integral membrane proteins within


9.5.43

Cell Adhesion

the plane of the cell membrane. The freedomof lateral movement allows the induction oflarge clusters of these molecules at the cellsurface by cross-linking their extracellularmoieties with antibodies, so-called capping.Molecules that undergo (direct or indirect) in-teractions with the capped molecules in theplane of the cell membrane are co-clustered andexhibit co-capping when examined by im-munofluorescence microscopy.

Antibody-induced co-capping has been anestablished technique, in particular in the fieldof immunology, for the last decades and wasinstrumental for the detection of many interac-tions of leukocyte surface molecules that areinvolved in antigen presentation and recogni-tion (Rojo et al., 1989; Zhou et al., 1993). Theco-capping approach allows detection of inter-actions between membrane proteins at the levelof individual intact cells. The availability ofnovel fluorescent dyes allows the detection ofthree or four different fluorochromes simulta-neously. This allows the examination of thecellular distribution of, for example, effectormolecules of signal transduction or cytoskele-ton components as a function of co-clusteringof cell surface proteins by a combination ofclassical co-capping protocols with intracellu-lar immunofluorescence staining.

Classical biochemical techniques, for exam-ple co-immunoprecipitation or chemical cross-linking (see Basic Protocol 5), require a homo-geneous sample. In contrast, co-capping stud-ies examine interactions between proteins at thelevel of individual cells and can, therefore, beperformed on heterogeneous samples, likemixed primary cell cultures derived from ani-mal tissue, if appropriate markers are availablethat allow one to distinguish between differentcell types. A further major advantage is therequirement for only a limited number of cellsfor co-capping studies. This opens the possibil-ity to study specific, rare cell types, which canbe isolated only in small quantities. A powerfulcombination is represented by the prior isola-tion of a specific cell type, e.g., by fluorescence-activated cell sorting (FACS), using appropriatecombinations of cell surface markers with sub-sequent co-capping studies.

The crucial prerequisite of the co-cappingapproach is the availability of suitable antibod-ies. This limits application of the technique toalready described molecules against which an-tibodies are available. In addition, the antibod-ies used must meet the following criteria, (1)absence of any cross-reaction with other cellsurface proteins on the cells of interest; and (2)

no interference of antibody binding with theinteraction that is studied (this point often rep-resents an unknown factor that may influencethe result). In addition, the two primary anti-bodies used must be raised in two differentspecies.


Preparation of IgSF-CAMsFor the functional analysis of an IgSF-

CAM, it is extremely important to have a highlypurified protein sample. Due to the high com-plexity of their interaction pattern, a contami-nation of the protein sample to be analyzed byanother IgSF-CAM could confound the resultsof binding assays (see Basic Protocols 2 and 3)or neurite outgrowth assays (see Basic Protocol7). For this reason, protein samples purifiedwith affinity columns should always be ana-lyzed by SDS-PAGE followed by silver stain-ing to confirm the absence of contaminatingproteins. When possible contaminants areknown, for instance based on their high expres-sion level in the tissue used as a source for thepurification, or when the absence of a particularIgSF-CAM is important for the experimentalprocedure, their absence can best be docu-mented by immunoblot analysis of the purifiedfractions. As a quick alternative, dot blots de-scribed in Support Protocol 4 can be used. Ifcontaminants are found, the following possi-bilities should be considered.

Affinity column was not rinsed properly af-ter loading or has run dry. Make sure absor-bance is back to baseline before starting elution.Take an aliquot from the wash fraction to con-firm the absence of protein. It is extremelyimportant that the column never runs dry.

Antibody used to make column is not specificor antibody is denatured because column is tooold or was not stored properly. When the qualityof the purified protein samples decreases, it istime to prepare a new affinity column. It ispossible that the antibody is very sensitive tohigh pH conditions used for elution. Try anelution buffer with low pH (0.1 M glycine/HCl,pH 2.7) as an alternative. Change concentrationand/or type of detergent used.

Because of the large number of IgSF-CAMsexpressed in the nervous system and based onthe enormous complexity of their interactions,the specificity of the antibodies used for func-tion-blocking assays and the purity of IgSF-CAM preparations have to be tested carefully.It is important to include appropriate controls,


9.5.44



such as the use of preimmune IgG purifiedaccording to the same protocols as the specificIgGs to rule out toxicity of the antibody prepa-rations. Assess the dose-dependence of the ef-fect. A very convincing control for the speci-ficity of the assay is the absence of an effect inthe presence of a specific antibody against an-other IgSF-CAM that is expressed by the samecell.

TransfectionThe most critical parameter determining the

efficiency of the calcium phosphate protocoldescribed is the 293 cells that are used. Cells oflow passage numbers (not more than 40 pas-sages) exhibit a much higher transfection effi-ciency than later passages.

The formation of the precipitate prior to theaddition to cells is mainly influenced by thefollowing parameters: concentrations of cal-cium, phosphate, and DNA, the nature of theDNA construct, pH, temperature, and time ofincubation. The calcium and phosphate con-centrations given in this protocol are optimizedfor this system. Optimal DNA concentrationdepends on the construct and must be deter-mined for each system individually. A goodstarting point for optimizations when dealingwith medium-sized plasmids of 5 to 10 kb is 40to 80 µg DNA. Plasmids of different sizes varyconsiderably in behavior in calcium phosphatetransfection. The protocol described works bestfor average sized expression plasmids (5 to 10kb). Very large plasmids (>30 kb) frequentlyresult in the formation of much larger precipi-tates, reducing the efficiency of transfectionconsiderably. A reaction at pH 7.0, room tem-perature (20° to 25°C), and 1 min reaction timeare good starting conditions for optimizations.The formation of precipitate after mixing CaCl2solution with HBS solution can be monitoredby measuring the absorbance at 320 nm (e.g.,for the testing of new batches of solutions).

After addition to the cells, the stability of theDNA-containing calcium phosphate particlesis the most critical factor for the efficiency oftransfection. One source of instability is a re-duction in medium pH due to the metabolicactivity of cells. It is therefore important toinclude 10 mM HEPES (final concentration) inthe 293 cell culture medium. The reduction ofthe CO2 partial pressure to 3% is another optionto raise the pH value, if necessary. Increasedtime of exposure to the precipitate can furtherenhance the efficiency of transfection. An in-cubation time of 4 to 6 hr given in the protocolis a good starting point and can be extended up

to 24 hr in cases where no cytotoxicity is ob-served. However, the combination of exposureto the precipitate with an osmotic shock, e.g.,by adding 10% (w/v) glycerol to the cell culturemedium, as suggested by some authors for thecalcium phosphate transfection of Chinesehamster ovary (CHO) cells, is not recom-mended for 293 cells.

ImmunoblottingNo positive dots. The concentration of the

protein of interest may be below the detectionlimit. Use repeated applications of protein so-lution to the nitrocellulose membrane. Applythe purified protein onto the membrane as apositive control. If the total protein concentra-tion of the solution applied to the membrane ishigh, even repeated applications of protein so-lution to the membrane may not give a satisfac-tory result.

High background. Make sure that the vol-umes of protein solution applied to the mem-brane are small. Let the dot dry before the nextstep. Wash membranes more thoroughly.Change the blocking solution and/or blockingtime. For instance, try blocking with serumfrom the same species that was used to raise thesecondary antibody.

Microsphere assaysFalse negative results in the bead aggrega-

tion assay. Coupling reaction results in proteinorientation on the bead surface in a way thatbinding sites are not accessible. Alternatively,proteins could not be physiologically activeafter purification (e.g., elution after immunoaf-finity column).

False positive results in the bead aggrega-tion assay. Protein preparations that are notpure and contain another CAM could result infalse positive binding results. Furthermore, ifeach of the two test proteins has homophilicbinding properties, weak unspecific aggrega-tion of preformed homophilic aggregates couldresult in false positive results with respect toheterophilic interactions. Therefore, check forunevenly distributed beads in mixed aggregatesunder the microscope (bead distribution in ag-gregates is not detectable in a flow cytometer).

Discrepancies between results of bead andcell aggregation assays. The orientation of pro-teins on the beads is likely to be random andnot uniform as in a biological membrane. Thishas to be considered when interpreting andcomparing the results with the ones of the cellaggregation assay. Furthermore, the orientationproblem has to be taken into account when


9.5.45

Cell Adhesion

analyzing cis- and trans-interactions (Kuhn etal., 1991; Buchstaller et al., 1996; Rader et al.,1996; Sonderegger et al., 1998).

Orientation of proteins on beads can be bet-ter controlled when using proteins with do-mains or tags that tightly bind to a linker proteinthat is coupled first to the microsphere. For ex-ample, recombinant Fc-fusion proteins can becoated on protein A-conjugated beads in an ori-ented manner. One has to consider that this link-age includes a noncovalent protein interaction.

Trans-interactionsThe stably transfected myeloma cell clones

may differ from the parental myeloma cell linein more than only the expression of the IgSF-CAM. The upregulation of endogenous celladhesion molecules, such as integrins andIgSF-CAMs, can result in myeloma cell aggre-gation (Kawano et al., 1991). It is thereforeessential to run a series of control experimentsthat address the question whether any cell ag-gregation is caused by a trans-interaction of theexpressed IgSF-CAMs or by other factors. Forthis, follow these guidelines:

1. Confirm the functional surface expres-sion of the IgSF-CAM using, for example,monoclonal antibodies directed against confor-mational epitopes.

2. Each aggregation assay should be per-formed in duplicate with cross-wise exchangeof the fluorescent dyes.

3. Myeloma cell clones that express anIgSF-CAM should not form mixed aggregateswith the parental myeloma cell line.

4. Independent myeloma cell clones thatexpress the same IgSF-CAM should give thesame aggregation pattern.

5. Independent myeloma cell clones thatexpress different quantities of the same IgSF-CAM should be analyzed for a correlation be-tween expression and aggregation.

6. Pre-incubation with polyclonal Fab di-rected to the IgSF-CAM should prevent aggre-gation.

7. Pre-incubation with an enzyme that re-moves the IgSF-CAM selectively from the sur-face should prevent aggregation. A very usefulenzyme for this is phosphatidylinositol-spe-cific phospholipase C, which selectivelycleaves glycosyl-phosphatidylinositol-an-chored proteins.

Chemical cross-linkingThe application of cross-linking reagents on

intact, live cells may result in possible artifactsdue to the perturbation of the architecture of the

cell membrane by the chemicals. Very often,the reagents are used at millimolar or higherconcentrations (0.1 to 10 mM) and must bedissolved in organic solvents prior to use. It istherefore very important to assure that the re-agents by themselves and especially organicsolvents, if used, do not affect the structuralintegrity and viability of the cells tested. Water-soluble reagents like BS3, Sulfo-DST, andDTSSP that carry sulfonyl groups can be usedto circumvent the solvent problem.

Successful detection of cis-interactions be-tween IgSF-CAMs requires a high specificityof chemical cross-linking on the one hand, andsufficient yields of cross-linked materials onthe other hand, for subsequent biochemicalcharacterization of cross-linked partners. Asdescribed above, the specificity and efficiencyof the cross-linking reaction is determined bythe following parameters: length of the spacerseparating the reactive groups; chemical reac-tivity of the functional groups of the cross-link-ing reagent; concentration of cross-linking re-agent; and reaction time.

Cross-linking reagents with short spacersbetween the reactive groups such as DST andSulfo-DST restrict chemical coupling toclosely associated molecules and are thereforepreferable for the detection of cis-complexesbetween membrane proteins. However, due tothe spatial proximity of their functional groups,these reagents exhibit an enhanced tendency forintramolecular cross-linking, i.e., coupling ly-sine side chains of the same molecule with eachother, which may result in very low yields ofcross-linked material. The application of re-agents with longer spacers between the reactivegroups such as BS3 and DTSSP generally re-sults in higher yields, but bears the risk ofunspecific reactions. As previously discussed,the chemical reactivity of the functional groupspresent in commercially available cross-link-ing reagents range from highly selective, likethe N-succinimidyl group, to rather unselectivephotoactivated groups, like aryl azides, whichgenerate highly reactive, unstable intermedi-ates that undergo reactions with a wide varietyof chemical structures within a protein. Al-though the N-succinimidyl group is most fre-quently used for modifications and cross-link-ing of cell surface proteins, its selective reac-tivity with primary amino groups (mainlylysine side chains) excludes this class of cross-linkers from extended hydrophobic interfacesthrough which IgSF-CAMs may interact. Uponphotoactivation, reagents containing aryl azidegroups exhibit a high reactivity towards ali-


9.5.46



phatic hydrocarbon groups, like RCH2R andR2CHR, that allows cross-linking within astrongly hydrophobic environment. However,the high reactivity and low selectivity of pho-toactivated aryl azides frequently results in ahigh degree of unspecific cross-linking, notonly among proteins, but also extensive pro-tein-lipid cross-linking. For the detection ofspecific interactions among the closely associ-ated proteins on the surface of intact cells, thesereagents are, therefore, not recommended asfirst choice, but represent an option in caseswhere no cross-linking products are obtainedusing more specific reagents.

Optimal concentrations of cross-linking re-agents must be evaluated empirically for everyexperimental system. A more detailed discus-sion of the chemical background can be foundin Lomant and Fairbanks (1976), Lewis et al.(1977), and Smith et al. (1978). For homobi-functional N-succinimidyl derivatives, likeDST and DSSP, optimal concentrations forcross-linking on intact cells are consistently inthe range of 0.1 to 10 mM in published proto-cols. This range of concentrations represents agood starting point for optimizations. Cross-linking reagents with higher reactivity, like e.g.,aryl azides, are generally applied in much lowerconcentrations, usually between 10 µM and 1mM, for cross-linking on intact cells.

A further critical parameter is reaction time.Longer reaction times generally result in betteryields of cross-linked products, but also in moreunspecific reactions. Using homobifunctionalN-succinimidyl reagents, quenching of thecross-linking reaction after 0, 5, 15, and 45 minresults frequently in a “kinetic profile” of theprocess. Specifically, a different pattern ofcross-linked products is observed during thetime course of the cross-linking reaction. Thisreflects the tendency of many membrane mole-cules, such as IgSF-CAMs, to form oligomericor even multimeric aggregates in the mem-branes of living cells. The appearance of initialcross-linked molecules, after a few minutes, isoften followed by the appearance of furthercross-linked complexes with higher molecularmasses, generated from the coupling of theinitial complexes with additional molecules.Initial cross-linked products that are generatedwithin the first minutes of the reaction maycorrespond to the first assembly units fromwhich larger oligomeric or multimeric com-plexes are formed. The “kinetic profile” of thecross-linking reaction may, therefore, givesome information about the nature of the com-

plexes or aggregates formed by molecules likeIgSF-CAMs.

Co-cappingThe demonstration of a cis-interaction by

antibody-induced co-capping of cell surfacemolecules critically depends on many factors.To allow the induction of caps by antibodycross-linking and co-capping mediated by acis-interaction, the molecules of interest musthave a minimal lateral mobility within the planeof the cell membrane. In addition to mobility,the relative stoichiometry of the molecules ofinterest is critical for the detection of co-cap-ping after antibody-induced capping. Ideally,comparable levels of expression at the cellsurface allow reciprocal co-capping of twomolecules that interact with each other in ciswith sufficient affinity. However, largestoichiometric excess of one molecule due todifferent expression levels results in asymmet-ric results in reciprocal co-capping experi-ments; capping of the more abundant compo-nent is followed by clearly detectable co-cap-ping of the less abundant molecule. In contrast,capping of the molecule expressed at lowerlevel results in only partial co-capping of themore abundant component. For the detectionof partial co-capping, confocal laser scanningmicroscopy is a powerful technique that allowsthe reliable detection of locally enhanced fluo-rescence signals (co-capping) versus a rela-tively high homogeneous background signal(uncapped monomers of the molecule presentin stoichiometric excess).

In addition to the factors described above,the availability of specific primary antibodiesagainst the molecules of interest, raised in dif-ferent species, is an essential prerequisite forco-capping experiments. For antibody-inducedcapping, polyclonal or monoclonal antibodiescan be used. Polyclonal antibodies are able tocross-link the cell surface antigen to some ex-tent in the absence of secondary antibody,whereas in the case of monoclonal primaryantibodies, cross-linking of bound primary an-tibody with the secondary antibody is requiredfor the induction of caps. The most criticalfactors of every co-capping experiment is thespecificity of the primary antibodies used. Amajor source of experimental artifacts is thepotential of cross-reactivity of primary anti-bodies, either between the molecules of interestor with other, unidentified molecules expressedon the cell. It is therefore of pivotal importanceto exclude any cross-reactivity under the con-ditions (antibody concentrations, temperature,


9.5.47

Cell Adhesion

incubation times, etc.) used for co-capping andcounter-staining. It is not recommended to testfor cross-reactivity with other immunochemi-cal techniques like immuno-blot or ELISA,because the antigens are presented in a differentform on nitrocellulose and on plastic surfacesthan on live cells. The absence of cross-reactiv-ity of the partially or totally denatured proteinspresent in such immunochemical assays does,therefore, not necessarily exclude cross-reac-tivity of the native proteins present on the livecells under the conditions used for co-cappingexperiments. In situations where the proteinsof interest are co-expressed in recombinantform in a cell type that does not normallyexpress them, the test for cross-reactivity isstraightforward. Specificity of detection can bechecked by immunostaining of single transfec-tants and mock-transfected cells (as a negativecontrol) with both antibodies (see, e.g., Buch-staller et al., 1996; Kunz et al., 1998). It shouldbe noted that cross-reactivity of antibodies is aphenomenon that depends on the antibody con-centrations used. Even in cases where no cross-reactivity is reported in the literature (e.g.,based on experience with standard protocols forimmunofluorescence), cross-reactivity mayoccur at the higher antibody concentrations thatare normally applied in co-capping experi-ments. Cross-reactivity of the secondary anti-bodies represents only a minor problem, sincehighly specific preparations of fluorochrome-labeled secondary antibodies against a widevariety of species are commercially available.The use of phylogenetically more distant spe-cies is desirable since the potential of cross-re-activity between the secondary antibodies islower. Combinations that are frequently docu-mented in the literature are mouse/rabbit,mouse/goat (or sheep), rabbit/goat (or sheep).A considerable risk of cross-reactivity existsespecially for the combination mouse/rat. Inthis case the choice of secondary antibodies hasto be made with care.

Several controls must be included to ensurespecificity of antibody-induced capping. Theuse of preimmune serum, or purified IgG frompreimmune serum, is an essential negative con-trol in cases where complete sera or total IgGfractions, respectively, are used as a source ofprimary antibodies. Additional controls shouldinclude the detection of unrelated moleculesexpressed by the cell of interest that are notexpected to co-distribute with one of the mole-cules tested for cis-interaction.

Apart from false-positive results due tocross-reactivity of antibodies, false-negative

results can be due to potential interference ofantibody binding with the interaction betweenthe molecules of interest. Polyclonal antibodiesdirected against a variety of epitopes can per-turb molecular interactions. It is thereforeworthwhile to test several different antibodies,polyclonal as well as monoclonal, if available.The potential interference of antibody bindingwith the interaction between the molecules ofinterest can be prevented by using heterolo-gously expressed recombinant proteins. Spe-cifically, different N-terminal peptide tags, likethe myc-tag or the influenza hemagglutinin-tagcan be introduced by molecular cloning into thepolypeptide sequences of the studied proteins.Subsequent capping with antibodies specificfor the N-terminally localized tag sequenceswill reduce the risk of interference with a bind-ing site on the surface of the molecule.

It should always be kept in mind, that thedetection of antibody-induced co-capping be-tween two molecules is no proof of a directmolecular interaction between the two compo-nents. In order to demonstrate such a directbinding, additional experimental techniques,like chemical cross-linking (see Basic Protocol5) or biochemical binding assays using iso-lated, purified proteins (see Basic Protocol 2)are required.

Neurite outgrowthMany IgSF-CAMs have a neurite out-

growth-promoting activity, therefore, neuritegrowth assays are widely used for functionalanalysis. The preparation of neuronal culturestakes some practice and should best be learnedin a laboratory where culture techniques areestablished. The reproducibility of the culturesis very important to get results. Keep in mindthat the growth and the morphology of neuritesdepend on the substrate and the medium. As thequality and the components of serum differconsiderably from batch to batch, the lot ofserum used should not be changed duringanalysis. For best reproducibility, the use of achemically defined, serum-free medium is rec-ommended. All the solutions and the purifiedproteins to be tested for their neurite outgrowth-promoting activity have to be of high quality.Especially, the absence of endotoxins is ex-tremely important for the survival of neurons.Make sure that detergents are removed care-fully from protein solutions before using themin tissue culture.

The growth characteristics of neurites areage-dependent. In particular, the dependenceon specific growth factors may change dramati-


9.5.48



cally. Therefore, it is essential not to mix dif-ferent ages of tissue. Similarly, the response ofneurons to different substrates may be species-specific. Remember that neurite length is notthe only criterion that can be assessed in neuritegrowth assays. Closely monitor neurite andgrowth cone morphologies and branching pat-terns.

Anticipated Results

TransfectionFor transient transfections with the protocol

described, reporter constructs can be used toassess the efficiency of transfection and calcu-late the percentage of transfected cells. Usingreporter constructs that express green fluores-cent protein (GFP) as a reporter under thecontrol of the human cytomegalovirus (CMV)immediate early promoter (plasmid size of 5 to10 kb, DNA amounts for transfection between40 and 80 µg), the authors repeatedly observedtransfection efficiencies of 30% to 40% basedon the detection of GFP expression 24 to 48 hrafter transfection. Expression of recombinantprotein under the control of strong viral pro-moters lasts, generally, for 48 to 96 hr aftertransfection.

Microsphere assaysWith the appropriate controls, all the proto-

cols described here that involve microsphereand cell aggregation techniques are powerfulmethods to study binding properties of IgSF-CAMs. Furthermore, these assays are not verytime-consuming and enable processing of sev-eral tests in parallel.

Trans-interactionsMyeloma cell clones that were stably trans-

fected to express IgSF-CAMs form aggregateswhen the IgSF-CAMs interact in trans. Homo-philic trans-interactions are indicated by aggre-gates of cells that express the same IgSF-CAM.Heterophilic trans-interactions are indicatedwhen two populations of cells that expressdistinct IgSF-CAMs form mixed aggregates.

Transfection-protoplast fusionOn average, 100 clones are obtained per

96-well tissue culture plate, i.e., 500 clones per5 × 106 transfected myeloma cells. Thus, thetransfection efficiency is in the range of 1 ×10−4. Even with an optimized electroporationprocedure, the transfection efficiency was ∼20times lower (Rader et al., 1993). While themajority of myeloma cell clones expresses

moderate amounts of the IgSF-CAM, a smallpercentage typically reveals very high expres-sion. Thus, the higher the transfection effi-ciency, the higher becomes the likelihood toobtain a myeloma cell clone with very highexpression. This makes protoplast fusion themethod of choice for the transfection ofmyeloma cells.

Chemical cross-linkingDuring optimization of the cross-linking

protocol, the emphasis should be placed on thespecificity of the reaction. As described, thereis generally an inverse relationship betweenspecificity of a cross-linking reaction and itsefficiency. A protocol that ensures a high degreeof specificity often has the drawback of lowyields of cross-linked material. Based on pub-lished results and experience in the authors’laboratory, yields of cross-linking protocols,like the one described, range from 0.1% to 1%,corresponding to a few hundred nanograms ofcross-linked material from a reaction per-formed on 106 cells. This amount of protein isnormally sufficient for immunochemical char-acterization of the cross-linked molecules, e.g.,by immunoblot analysis or re-immunoprecipi-tation (Buchstaller et al., 1996; Kunz et al.,1998).

The appearance of only one or a few com-plexes of a molecule of interest indicates somedegree of specificity of the reaction. The cross-linked complexes isolated by immunoprecipi-tation can be separated by SDS-PAGE (ideallytwo-dimensional; UNIT 6.4). The detection ofpresumed binding partners in the cross-linkedcomplexes by immunoblot analysis should al-ways include controls, that is the detection of amembrane protein present in the cell used forcross-linking that is not expected to associatewith the molecule of interest.

Co-cappingAntibody-induced co-capping can be ob-

served between molecules that undergo director indirect cis-interactions. Very clear resultscan be obtained in cases where the two compo-nents are expressed at comparable levels anddirectly interact with each other with relativehigh affinity, as demonstrated in the exampleshown in Figure 9.5.3. Co-capping of moleculeA (NgCAM) with molecule B (axonin-1) wasstudied on stably double-transfected CV-1cells. Capping of molecule A (NgCAM), in-duced by the subsequent incubation with amouse monoclonal primary and a rabbit-antimouse secondary antibody (Fig. 9.5.3A), re-


9.5.49

Cell Adhesion

sults in extensive co-capping of molecule B(axonin-1), as shown by counter-staining withgoat anti-B (axonin-1) Fab fragments on thesame cell (Fig. 9.5.3C). As expected, no cap-ping or co-capping is detected in the absenceof primary antibody against molecule A(NgCAM).

Time Considerations

Preparation of affinity columnThe preparation of an affinity column takes

1 working day. Because affinity columns haveto be loaded slowly, it is convenient to load largevolumes of protein solution overnight to havethe column ready for elution the following day.However, ensure that the column never runsdry.

TransfectionThe entire transfection procedure can be

performed in <1 hr for up to 12 samples.

Trans-interactionsStable transfections of myeloma cells in-

cluding selection, subcloning, and analysis re-quire 4 to 6 weeks. Myeloma cell aggregationassays can be completed in <1 day.

Chemical cross-linkingThe entire procedure can be performed

within 2 days. The cross-linking reaction, in-cluding the making of the cell lysates, can becarried out within 3 to 4 hr. The lysates can bestored for several weeks at −20°C. Immunopre-cipitation can be performed either with an in-cubation of 4 hr with the first antibody or withan incubation overnight.

Co-cappingThe entire procedure can be performed in 4

to 5 hr. Incubation periods given here for anti-body-induced capping are based on the authors’own optimized and published protocols. Thetime periods given for counter-staining can beextended for the primary antibody from 1 hr atroom temperature to overnight at 4°C, if desiredor necessary for higher sensitivity. The protocolcan be interrupted after step 11 and 16 and thefixed coverslips stored at 4°C under light pro-tection for up to 1 day before the subsequentsteps are carried out.

Neurite outgrowthThe time required for neurite growth assays

depends on the type of neurons used. Dissoci-ated dorsal root ganglia neurons, for instance,will extend long neurites on many IgSF-CAMs

Figure 9.5.3 Antibody-induced co-capping of axonin-1 with NgCAM on stably double-transfected CV-1 cells. In double-transfected CV-1 cells, NgCAM (A) and axonin-1 (C) were randomly distributed. Capping of NgCAM was induced by a mousemonoclonal antibody and a rabbit anti-mouse IgG. NgCAM caps were detected by a Texas Red–labeled donkey-anti-rabbitIgG (B). The distribution of axonin-1 was detected by counter-staining with goat anti-axonin-1 Fab fragments and aFITC-labeled donkey anti-goat IgG (D). For examination of the cells, a confocal laser-scanning microscope equipped withan argon/krypton laser was used. Texas Red was detected using the 568-nm band-pass excitation filter (A,B) and FITC withthe 488-nm band-pass excitation filter (C,D), minimizing cross-talk between the two fluorochromes.


9.5.50



in <24 hr. DRG explants may take an additionalday to reach their full length. Coating of IgSF-CAMs as a substrate is best done immediatelybefore neurons or explants are plated, whereasdishes coated with poly-D-lysine or collagencan be stored. Dishes coated with laminin orIgSF-CAMs should not be stored and must notbe dry.

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Wigler, M., Silverstein, S., Lee, L-S., Pellicer, A.,Cheng, Y., and Axel, R. 1977. Transfer of purifiedherpes virus thymidine kinase gene to culturedmouse cells. Cell 11:223-232.

Wood, C.L. and O’Dorisio, M.S. 1985. Covalentcross-linking of vasoactive intestinal polypep-tide to its receptors on intact human lym-phoblasts. J. Biol. Chem. 260:1243-1247.

Zhou, M.-J., Todd, R.F., van de Winkel, J.G.J., andPetty, H.R. 1993. Cocapping of the leukoadhesinmolecules complement receptor type 3 and lym-phocyte function-associated antigen-1 with Fcgamma receptor III on human neutrophils. Pos-sible role of lectin-like interactions. J. Immunol.150:3030-3041.

Contributed by Peter SondereggerUniversity of ZurichZurich, Switzerland

Stefan Kunz and Christoph RaderThe Scripps Research InstituteLa Jolla, California

Daniel M. SuterYale UniversityNew Haven, Connecticut

Esther T. StoeckliUniversity of BaselBasel, Switzerland


9.5.52



UNIT 9.6Measurement of Adhesion Under FlowConditions

This unit describes the analysis of dynamic cell adhesion using a flow chamber assay. Theflow chamber enables the researcher to reconstruct cell systems in the presence of shearstress to assay adhesion under well-defined forces. These assays are most commonly usedto study leukocyte adhesion, either to cultured endothelial cell monolayers or to purifiedsubstrates, simulating physiological interactions of leukocytes with endothelial cells. Theassay described (Basic Protocol 1) utilizes commercially available parallel-plate flowchambers, in which cells are introduced under conditions of laminar flow between twoflat surfaces, allowing for visualization of dynamic adhesion on a microscope (Lawrenceand Springer, 1991). Flow cell assays are particularly useful to investigate adhesive eventsthat occur very rapidly, on a time scale shorter than those assayed by static assays (Alonet al., 1995; Smith et al., 1999). Some adhesive events occur only in the presence of shearand, thus, cannot be characterized under static conditions (Finger et al., 1996; Lawrenceet al., 1997). In addition, the flow cell assay allows visualization of the subprocesses ofadhesion, including rolling, firm arrest, adhesion strengthening, spreading, and migration(Cinamon et al., 2001). Methods of data analysis are also discussed (Basic Protocol 2).

This assay can be also be used to characterize transient adhesive events or adhesionstrengthening even for cells that do not normally experience shear stress, because contacttime between cells and substrates and anti-adhesive forces can be closely regulated bystopping and starting the flow (Kassner et al., 1995). Flow chamber assays are also usefulfor measuring bacterial adhesion under flow (Mohamed et al., 2000; Poelstra et al., 2000).

BASICPROTOCOL 1

FLOW ASSAY FOR CELL ADHESION

Commercial flow chambers incorporate the surface of either a culture dish or a coverslipas one of the two parallel plates between which laminar flow occurs. This surface shouldbe coated with either endothelial cells or purified extracellular matrix proteins as anadhesive substrate.

Although several flow chambers are available, the same basic methods are used to conductflow cell adhesion experiments regardless of the particular flow chamber apparatus used.Until recently, investigators often constructed their own laminar flow chambers, butwith the advent of relatively inexpensive commercial models, this is not worthwhileunless special characteristics are required. The following method usually assumes thata GlycoTech flow chamber is used, but this protocol can easily be adapted for use withother chambers. Simply modify the assembly procedure according to the manufacturer’sinstructions and use cell- or ligand-coated glass coverslips instead of coated dishes ifrequired.

NOTE: All culture incubations should be performed in a humidified 37◦C, 5% CO2

incubator unless otherwise specified.

Materials

Substrate coating solutions:Fibronectin, gelatin, or other extracellular matrix proteins, either alone or in

combination, for cell monolayers only10 μg/ml matrix protein, such as fibronectin or collagen, in PBS (APPENDIX 2A),

pH 7.4, for dishes and coverslips coated with purified ligand only

Contributed by Dennis F. KucikCurrent Protocols in Cell Biology (2009) 9.6.1-9.6.10Copyright C© 2009 by John Wiley and Sons, Inc.

Cell Adhesion

9.6.1

Supplement 43

Measurement ofAdhesion UnderFlow Conditions

9.6.2


Cultured endothelial cells (UNIT 1.1) growing as a confluent monolayer culture intissue culture dish or on a coverslip of a size appropriate for the chamber used

1% (w/v) BSA in PBS (APPENDIX 2A), pH 7.4, for dishes and coverslips coated withpurified ligand only

PBS (APPENDIX 2A), pH 7.4, for dishes and coverslips coated with purified ligandonly

100 μg/ml poly-L-lysine (PLL; e.g., Sigma), for coverslips coated with purifiedligand only

1% (v/v) glutaraldehyde, for coverslips coated with purified ligand onlyLeukocytes or other cells of interestHBSS (APPENDIX 2A), room temperature and 37◦C2 mM 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, acetoxymethyl ester

(BCECF-AM; Molecular Probes) in DMSO

35-mm plastic tissue culture dishes for GlycoTech chamber or glass coverslips of asize appropriate for the flow chambers that require them, sterilized byautoclaving, UV light, or flaming with ethanol

35-mm tissue culture dishes or 6-well tissue culture plates, for glass coverslips onlyPermanent marker or diamond stylus, for dishes coated with purified ligand only25-ml syringesProgrammable syringe pumpFlow chamber (e.g., GlycoTech) and appropriate tubingInverted phase-contrast microscope with low-power (∼10 to 20×) objective,

fluorescence optics (recommended but not required), and stage incubator set to37◦C

Video camera (e.g., CCD-300T-RC; Dage-MTI) and other recording equipment(e.g., VCR, television monitor, video cables)

Prepare substrates

For cell monolayer preparation

1a. Incubate a 35-mm sterile plastic tissue culture dish or glass coverslip with 1 ml sub-strate coating solution of fibronectin, gelatin, or other extracellular matrix proteins,either alone or in combination (Table 9.6.1) for 30 min at 37◦C.

There are a number of commercially available flow chambers that require glass coverslipsof different sizes. In general, only a small portion of the surface area of the coverslip isused for the assay. The size of the coverslip required has more to do with the geometry ofthe chamber than the actual surface area used.

2a. Remove excess coating solution and, if using coverslip, place the coverslip in a35-mm dish or in the well of a 6-well tissue culture plate.

Table 9.6.1 Endothelial Cells and Culture Conditions

Cell typea Dish coatingb Plating density(cells/cm2)

Days to confluence

HUVEC 0.05% (w/v) gelatin

10 μg/ml fibronectin 5000 3-4

HAEC 0.05% (w/v) gelatin

10 μg/ml fibronectin 5000 3-4

MAEC 1% (w/v) gelatin 6000 8-10aAbbreviations: HAEC, human aortic endothelial cells; HUVEC, human umbilical vein endothelial cells; MAEC, mouseaortic endothelial cells.bThe buffer used for the coating solution is HBSS (Hanks’ balanced salt solution).

Cell Adhesion

9.6.3


3a. Add 2 ml endothelial cell suspension (see Table 9.6.1 for cell density). Culture at37◦C.

4a. Feed cells every 2 to 3 days with an appropriate medium until confluent. Continuewith step 9.

Coating density and the time to reach confluency depend on the cell type (Table 9.6.1).Optimal conditions for endothelial cell types other than those listed in Table 9.6.1 shouldbe worked out empirically. Higher seeding densities result in less time to confluency, butlower densities and more time generally result in a more uniform monolayer. Dependingon the cell type, cells are usually suitable for assays for 2 to 3 days after they reachconfluency.

Endothelial cell adhesion to tissue culture plastic is generally superior to adhesionto glass coverslips, even if both are coated with extracellular matrix materials. Opticalproperties of plastic, however, are inferior to glass, because of a relatively high fluorescentbackground. Birefringence of plastic can also be a problem if Nomarski imaging isused. The optical properties of plastic are, however, adequate for the level of resolutionneeded for visualization and analysis of adhesion under flow, and they can provide bettervisualization of rolling cells than, for example, ex vivo preparations of blood vessels.

Leukocytes interact more with endothelial cells that have been stimulated. Interleukin1 (IL-1), tumor necrosis factor α (TNF-α), and lipopolysaccharide (LPS) have beenconsidered good paradigms for pro-inflammatory mediators and are often used exper-imentally on cultured endothelial cells to simulate inflammation (Cines et al., 1998).Stimulation results in expression of a number of adhesion molecules, in addition to othereffects. Whether and how the endothelial cells should be stimulated will depend on thephysiologic conditions that are being mimicked.

For plastic dishes coated with purified ligand

1b. Use a permanent marker or diamond stylus to outline a 5-mm diameter area of asterile plastic tissue culture dish for coating.

If a diamond stylus is used, the scratches should be shallow enough that they do notprevent the flow chamber gaskets from sealing.

The size of the area covered depends less on the size of the dish or coverslip than on thearea of obseervation required. The authors find that a 5-mm diamerer circular area issufficient to provide several fields of view for independent measurements of cell-substrateadhesion.

2b. Add 25 μl of 10 μg/ml matrix protein in PBS to the marked area and incubate 1 hrat room temperature.

3b. Remove coating solution and add 1 ml of 1% BSA in PBS. Incubate 1 hr at roomtemperature.

4b. Remove BSA solution. Wash twice with 5 ml PBS each. Continue with step 9.

The coated dish can be stored for 1 day at 4◦C.

For glass coverslips coated with purified ligand

1c. Place sterile glass coverslip in a 35-mm tissue culture dish or in the well of a 6-welltissue culture plate.

2c. Add 0.2 ml of 100 μg/ml poly-L-lysine (PLL) to the dish. Incubate 10 min at roomtemperature.

The amount of PLL used should be scaled up as needed depending on the coverslip size(e.g., 0.5 ml should be used for a 22 × 22–mm coverslip). The authors routinely use PLLwith a molecular weight of 70 to 150 kDa.

3c. Wash coverslips three times with 5 ml PBS each.


9.6.4


4c. Add 1% glutaraldehyde and incubate 30 min at room temperature.

5c. Wash coverslips three times with 5 ml PBS each.

6c. Add 25 μl of 10 μg/ml matrix protein and incubate 1 hr at room temperature.

For a 22 × 22–mm coverslip, 100 μl coating solution should be used.

7c. Add enough 1% BSA in PBS to completely cover the coverslip to block. Incubate30 min at room temperature.

8c. Wash coverslip with 5 ml PBS. Add fresh PBS to keep moist until used for flowassay. Use within 1 day.

This protocol, in which matrix molecules are covalently linked to adsorbed PLL, resultsin tighter, more uniform binding of matrix materials to coverslips than direct adsorptionof matrix molecules to the glass. If, however, PLL needs to be avoided, direct adsorptionof matrix materials, as in the plastic dish protocol, may provide an adequate substrate.

Coverslips precoated with PLL are also commercially available, but the authors have nottried them.

Figure 9.6.1 Visualization of endothelial cells and leukocytes using (A) phase contrast and (B)fluorescent optics. Arrows in (A) indicate leukocytes. Fluorescent optics provide high-contrastimages that facilitate manual counting and are especially useful for computer tracking. Whenusing endothelial cells as the substrate, however, it is important to observe the system with phasecontrast or similar optics to assess the integrity of the monolayer before switching to fluorescence.

Cell Adhesion

9.6.5


Load cells with flourescent dye(optional)9. Wash leukocytes or other cells of interest in HBSS and then resuspend them at 1–2

× 106/ml in 25 ml HBSS.

Although rolling and adherent cells can be visualized without fluorescence, the increasein contrast afforded by labeling the perfused cells greatly facilitates observation andcounting (Figure 9.6.1) and is sometimes a requirement for object-tracking software.

10. Add 12.5 μl of 2 mM BCECF-AM and incubate 30 min at room temperature in thedark.

11. Wash cells three times with 35 ml HBSS each.

Carry out flow assay12. Resuspend leukocytes in prewarmed HBSS to a concentration of 0.5 × 106 cells/ml

Cell concentration should be high enough that sufficient events are observed in the fewminutes of recording, but not so high that cell behavior is dominated by collisions withother rolling or adherent cells. The authors find that 0.5 × 106 cells/ml works well. Thenumber of cells required will depend on the rate of flow and the length of observationneeded to gather sufficient data to answer the particular experimental question. Theauthors usually use 3–5 × 107 cells/experiment (including controls).

In general, it is a good idea to perform experiments at 37◦C (this necessitates a stagewarmer), although useful data can sometimes be obtained at room temperature, dependingon the experimental question.

13. Load leukocytes into a 25-ml syringe, if using the pushing method, or into a centrifugetube or other reservoir, if using the pulling method.

There are two ways to use a syringe pump to perfuse cells through a flow chamber. Thesyringe can be filled with cells and the pump can be used to push them through thechamber. Alternatively, some investigators prefer to attach the syringe to the outlet sideof the system to pull fluid and cells through the chamber.

14. Attach the tubing of a flow chamber to the syringe and remove any air bubbles fromthe system.

15. Assemble a flow chamber according to the manufacturer’s instructions, using atissue culture dish containing the monolayer of cultured endothelial cells (step 4a)or coated with purified ligand (step 4b or 8c). Again, work out any bubbles in thesystem.

Flow chambers come in a wide variety of sizes. Generally, the smaller the chamber, themore economical the experiment is in terms of cells used per experiment. The dimensionsof the GlycoTech flow chamber are determined by the size of the gasket used becausethe gasket forms the side walls of the chamber and also determines its height. GlycoTechsupplies four gaskets with its basic kit; these range in width from 0.25 to 1.0 cm in widthand in thicknesses ranging from 0.005 to 0.010 in. The length is fixed at 2 cm.

Increasing the dimensions of the flow chamber will decrease the wall shear stress fora given flow rate. This has the disadvantage of requiring more volume (and thus morecells) to be perfused per unit time for a given shear. It can be advantageous, however,to use a larger chamber when low shears are desired because the syringe pump flowrates are less accurate and more pulsatile as flow is decreased. A wider chamber hasthe further advantage of providing more observation area if many fields of view aredesired.

Significant shear stresses can be created during assembly, which can sometimes tearendothelial cell layers loose from the dish. Therefore, care must be taken to assemble thesystem slowly and gently.


9.6.6


16. Program the syringe pump to give the desired flow according to the manufacturer’sinstructions.

To determine the desired flow, flow rates must be converted to shear stresses. The man-ufacturer of the flow chamber will usually provide a handy conversion chart or table.Alternatively, shear stress can be calculated from the dimensions of the flow chamberand the rate of fluid flow (Lawrence et al., 1987). The shear stress desired will depend onthe physiological condition to be mimicked. For example, simulation of arterial flow willrequire higher shear rates than simulation of venous or postcapillary venule conditions.The authors usually do a range of shears to determine the shear dependence of anyphenomena observed. As a rule of thumb, physiologic shear stresses are typically thoughtto be in the range of 0.5 to 5.0 dynes/cm2.

17. Mount the flow chamber on an inverted phase-contrast microscope with a prewarmedstage incubator. Focus the system and adjust the optics, video camera, and any otherrecording equipment as needed.

If using endothelial cells, it is important to visualize the area of observation adequately toassess the integrity of the monolayer. Rips or other defects in the monolayer will confoundinterpretation of the results, because the data will reflect interactions of flowing cells notonly with endothelial cells but also with bare plastic or glass.

It is advisable to do a sample recording of a minute or so at this point and to play it backto be sure that all of the equipment is working properly.

18. Choose an area to observe, start the recording equipment, and begin the flow.

The authors record on sVHS videotape for later digitization. Images can also be captureddirectly to computer. This may be necessary if the camera used does not have a videooutput.

19. Record a few minutes of data at each shear rate.

The authors generally record ∼3 to 6 min of data at each shear rate from each area ofobservation. For preliminary experiments they use at least three or four shear rates. Oncethe optimal shear and the shear dependence are determined, one or two shear rates maybe sufficient. Considerations include the number of cells available (e.g., primary mousecells are more precious than cell lines) and any time dependence of treatment regimens.It is important to record diligently the points on the videotape where shear rates arechanged so that data can be interpreted when it is time to do the analysis.

20. Move to a new field of view and repeat steps 18 to 19. If using the pushing method,with the cells in the syringe, rotate the syringe 180◦ to compensate for any settling. Ifusing the pulling method, with the cells drawn from a tube or other reservoir, makecertain that the cells are still suspended.

Most flow systems are large enough to allow a number of areas of observation for eachpreparation. It is important to observe multiple areas to control for any peculiarities indensity of substrate coating or properties of endothelial cells. The number of fields toobserve for each preparation will again depend on the number of cells available, anytime dependence of treatment regimens, and the statistical power required.

21. Perform data analysis (see Basic Protocol 2).

BASICPROTOCOL 2

DATA ANALYSIS

Quantifying Adherent CellsThe raw data from flow cell adhesion experiments will be videos of rolling and firmlyadhering cells. The method of analysis will depend on the scientific questions to beanswered. A simple count of adherent cells per minute can be obtained by visual reviewof videotapes. Just choose an appropriate time period (e.g., 1 min) and roll the tape tocount the number of cells that become immobilized in the field of view during that time.

Cell Adhesion

9.6.7


Figure 9.6.2 Velocity profile for fluid flow in a parallel-plate laminar flow apparatus. Longer arrowscorrespond to higher velocities. The velocity profile is parabolic, with a maximum velocity midwaybetween the two plates and a slowest fluid flow near the plates.

It may be helpful to use two people to do this, one who counts and one who watches thetime. Because the number of cells recorded will depend on the size of the field observed,it should be normalized for the area of observation. Area can be calculated using a stagemicrometer, available from many scientific supply companies (e.g., Fisher), as a knownlength standard. A stage micrometer (also called a calibration plate) is a glass slide withaccurate distance scales etched into the surface. By imaging this size standard, the size(in microns) of the video image or monitor screen can be determined.

An important consideration for meaningful comparisons of experimental conditions isthat the number of cells perfused per minute must be the same under all experimentalconditions. This requires that cells are loaded into the syringe or reservoir at a consistentdensity and that cells are not allowed to settle out of suspension.

Quantifying Rolling CellsThe number of rolling cells per minute can also be determined by visual review, but caremust be taken in identification of rolling cells. By definition, under laminar flow therewill be a parabolic hydrodynamic velocity profile, with the lowest velocity of the fluidoccurring nearest the flow chamber wall (Figure 9.6.2). Thus, it may not be obvious whichcells are moving slowly because of their position in the flow field and which are retardedby adhesive interactions (Goldman et al., 1967). Whereas under some optical conditionsrolling cells can be distinguished by plane of focus, it is a good idea to calculate a criticalvelocity to verify that cells counted as rolling do indeed have adhesive interactions withligand. The critical velocity is an objective measure that has been developed to identifyrolling cells (Ley and Gaehtgens, 1991). A simple calculation for critical velocity isVc = βr γ, where Vc is the critical velocity, β is a drag factor, r is the radius of the cell,and γ is the shear rate. A reasonable estimate of β that can be used for cells in a flowchamber is 0.5. Cells moving more slowly than the critical velocity can be confirmed asbeing rolling cells. Thus, even if the experimental question deals only with the numberof rolling cells, and not their velocity, a few velocities should be measured to ensure thatwhat appears to be rolling cells are moving more slowly than the critical velocity and,thus, are indeed rolling. With some practice, though, rolling cells can often be identifiedvisually.

Rolling VelocitiesThe calculation of cell position in each digitized image can be done by comparison witha known size standard, again using a stage micrometer. A known length (in microns) canthen be marked on the monitor screen with a laboratory marker. With a known distance


9.6.8


established, velocities can be determined by simply counting the number of video framesrequired for a cell to travel this known distance. Alternatively, more accurate velocitydeterminations can be made using more sophisticated image processing software thatcan accurately localize objects in each image and convert pixels to distances. A varietyof commercial software packages are available. NIH Image, which is available online atno cost, will also track object motion (see Internet Resources).

COMMENTARY

Background InformationLeukocyte rolling and adhesion in vivo

has been observed microscopically for morethan 100 years (Cohnheim, 1889). More re-cently, investigators have used parallel-plateflow chambers to mimic the environment ofthe blood vessel under more controlled con-ditions. Much of what we know today aboutinflammation and leukocyte homing has beenlearned by examining leukocyte interactionswith either endothelial cell monolayers or pu-rified ligands in flow chambers (Butcher, 1991;Rossiter et al., 1997; Smith, 2000; Orselloet al., 2001). In addition, adhesion assays us-ing flow chambers have provided importantinformation about bacterial adhesion to sur-faces. Today, adhesion under flow can readilybe assayed economically using a variety ofcommercially available flow chambers. Whenmounted on a microscope and coupled to avideo camera and a VCR, simple analysis ofadhesion under shear is possible. More de-tailed analysis of rolling cell motion can beachieved by digitization of images coupledwith motion analysis software.

Commercially available flow chambers, ingeneral, work very well and make it easy toproduce conditions that mimic physiologicalcell interactions with well-defined and tightlycontrolled shear stresses. Increased use ofthese assays will provide a fuller understand-ing of physiologic cell adhesion than couldever be achieved with static adhesion assays.


Flow experiments require some diligenceto ensure that the system is working prop-erly. The experimenter should watch the mon-itor carefully during the experiment to be surethat the flow is smooth and that perfused cellconcentrations are uniform. Although a pro-grammable syringe pump is capable of pro-viding very steady perfusion, flow can becomepulsatile when the syringe is almost empty orif the syringe is not mounted securely. Othersources of variability in flow are leaks or bub-bles anywhere in the system. It is also a goodidea to isolate the system from vibration.

If fluorescent dyes are used to label per-fused leukocytes, it is a good idea to developa protocol that labels a high percentage ofthe cells (>97%), because unlabeled cells willlikely be missed during analysis, confoundingquantification of the number of cells interact-ing with the endothelium or substrate. Careshould be taken, however, not to overload thecells, because fluorescent dyes can be some-what toxic. Too much label interferes with cellmotility and may affect regulation of adhesionin general. The optimal dye-loading protocolcan be determined by varying both dye con-centration and incubation times and should beworked out for each new cell type.

When using object tracking software, theexperimenter should observe the trackingclosely. Although most available programs doa very good job and provide temporal reso-lution not obtainable by cruder methods, theyalso make occasional errors, briefly lockingonto an object other than the cell of inter-est. The operator should confirm visually foreach cell track that the software has faith-fully tracked the object of interest and has notmisidentified the cell at any point.

Uniformity and integrity of the substrateare also of vital importance. This is especiallytrue of endothelial cell monolayers. High shearforces associated with assembly of the cham-ber or with manual flushing of bubbles candislodge portions of the monolayer. Thus, toavoid mistaking leukocyte interactions withthe chamber itself for interactions with theendothelium, the importance of a visual in-spection of each recorded field of view cannotbe overemphasized. Purified protein substratespresent less of a problem, but care should betaken to ensure uniform distribution and den-sity of adsorbed matrix materials through useof good experimental technique.

CamerasThe authors recommend cameras with a

video (rather than digital) output, because stor-age of images is more economical on video-tape than on digital media. This allows theexperimenter to record continuously through-out the experiment. This can be an important

Cell Adhesion

9.6.9


consideration if one is working with valuableperfused cells (e.g., leukocytes purified fromgene-targeted mice). An economical record-ing system also encourages collection of themaximum amount of data from each experi-ment, avoiding the need to repeat experimentssimply to increase statistical power. In addi-tion, higher time resolution (30 frames/sec) ispossible with video than with most digital sys-tems. Images can be digitized later for com-puter analysis using a variety of commerciallyavailable systems.

If fluorescent cells are to be visualized, ahighly light-sensitive camera must be used.This is because when rolling cells are visu-alized, the time to acquire the image is lim-ited. Extended exposures cannot be used, asthe cells will appear blurry from their move-ment. Several camera companies offer cam-eras that are sensitive enough for this ap-plication, but the authors know of few withboth adequate sensitivity and video output.The authors have found the CCD-300T-RC(Dage-MTI) to work well. Equivalent camerasmay be available from other manufacturersas well.

It is also possible to do flow cell experi-ments without fluorescently labeling the cells.Although this results in less contrast betweenrolling cells and underlying endothelial cellsor substrate, the images can be perfectly ac-ceptable (Fig. 9.6.1). Imaging without fluo-rescence also avoids possible deleterious ef-fects of dye loading on the cells. In addition,the time required for each experiment is short-ened by the amount of time needed to load thecells with fluorescent dye, which may be use-ful if short-acting treatments are used. A ma-jor determinant of whether fluorescence canbe omitted is the quality of the microscopeoptics. Another factor to consider, however, iswhether any tracking software to be used de-pends on cell intensity for tracking motion. Al-though some programs can track objects basedon their size and shape, other packages requirethe tracked object to be brighter than its sur-roundings.

When imaging without fluorescence, it isespecially important to avoid fingerprints andother smudges on the culture dish or coverslipand other optical elements of the flow chamber,to avoid degrading the image.

Video recordingIt is recommended that video be recorded

using an sVHS, rather than a VHS (the typicalhome-recording format), VCR. Not only willthe sVHS spatial resolution be better, but the

recordings are more resistant to degradationwith repeated playback. Alternatively, imagescan be recorded directly to computer memoryor hard drive. With some cameras, this may bethe only option.

Image digitization and compression tech-nologies are progressing so rapidly that anyhardware recommendations are likely to be outof date within months. Therefore, it is recom-mended that the investigator research availableproducts when setting up a system. An impor-tant factor to consider is the amount of com-pression necessary with each system, becauseincreased compression can lead to decreasedimage quality and, consequently, a decrease inaccuracy of cell position determination.

Anticipated ResultsIn general. leukocytes perfused over stim-

ulated endothelial cells at physiological flowrates will be expected to interact with bothrolling and firm adhesion. The amount andtype of interaction will depend on the leuko-cyte type and its state of activation as wellas the endothlial cell type (venous versusarteerial, mouse versus human). Endothelialcells can be stimulated by TNF-α (e.g., 10to 100 ng/ml for 8 hr). This induces expres-sion of a number of adhesion molecules andshould dramatically increase adheive interac-tions with almost any leukocyte type. Hu-man umbilical vein endothelial cells (HUVEC;UNIT 2.3) have been used in a number of flowchamber adhesion studies (Munn et al., 1994;Macconi et al., 1995; Thelilmeier et al., 1999;Patel, 1999; Kaur et al., 2001), are widelyavailable, and make a good test system to val-idate experimental technique.

Time ConsiderationsThe time required to prepare a cul-

ture monolayer is 1 to 2 hr with severaldays required for cells to reach confluency(Table 9.6.1). Preparing a pure ligand substraterequires 5 to 6 hr for either culture dishes orcoverslips. The flow assay requires about 3 hrto do two cell types and three conditions, andat least 1 day, depending on the analysis per-formed is needed to analyze the data.

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Contributed by Dennis F. KucikUniversity of Alabama at BirminghamBirmingham, Alabama

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