8th SEMINARSEPARATION AND MEASUREMENT OF THE ACTIVITY OF IMMUNECOMPETENT CELLS

CELL SEPARATION

Physical isolation of the cells of interest from a heterogeneous population

Differences in the physical, biological or immunological properties of the cells are utilized to separate the cells. (Differences in cell surface receptor expression is often available – there is a possibility to further investigate the separated living cells).

physical – density, size cell biological – adherence, phagocytosis, sensitivity to the

medium immunological – antigen differences (surface marker)

Consideration taken to: purity, recovery, yield and viability of the cells

TWO SEPARATION STRATEGIES

Positive separation

Labeling and separation of

the cells of interest e.g. labeling a cell surface molecule by a

fluorescent antibody.

The cells become affected both by the separation environment and the antibodies bound to the receptors. The purity of the separation is generally high.

Negative separation

Labeled the unwanted cells (depletion)

The cells become affected only by the separation environment, hence this is the preferred strategy in functional examinations.

peripheral blood (or buffy coat)

pipetting cells on ficoll

centrifugation

separated cells

plasma

ficoll

Red blood cells

mononuclear cells

(PBMC)

Neutrophilgranulocytes

pipettig the „ring” containing the mononuclear

cells to a new tube to get rid of Ficoll

FICOLL-PAQUE DENSITY BASED CELL SEPARATION

(Nature Protocols http://www.nature.com/nprot/journal/v3/n6/images/nprot.2008.69-F1.jpg)

(from Google pictures)

Magnetic-Activated Cell Sorting (MACS)

paramagneticbead

antigen specific antibody

SEPARATION METHODS BASED ON THE IMMUNOLOGICAL PROPERTIES OF THE CELLS

MAG

NETM

AGN

ET

column

depleting or selecting

unlabeled cells(negative

separation)

Fluorescence-Activated Cell Sorting (FACS)

SEPARATION METHODS BASED ON THE IMMUNOLOGICAL PROPERTIES OF THE CELLS

Example:NKT cell separation (CD3/CD56)

NK cells

NKT cells

lymphocytes

blood sample

T cells

The fluid stream break up into droplets by the

vibration of the flow cell.

breakoff point

Laser

+ ----

+++

+++ +

+

--

---

vibration (nozzle orifice of the flow cell)

If the wanted cell reaches the breakoff point, the stream become charged for the short time of drop formation, so the formed drop become charged

charged deflection plate

charged deflection plate

++++++++

+

collection tube collection tube

waste

MEASURING THE ACTIVITY OF IMMUNECOMPETENT CELLS

PHAGOCYTIC CELLS – PHAGOCYTOSIS ASSAY

• Using killed pathogens (bacteria: E. coli, S. aureus; yeast: S. cerevisiae) labeled with different fluorophores

• Phagocytosis can be detected by fluorescent microscopy or by flow cytometry

The activation of lymphocytes by a specific antigen is hardly detectable (low numbers of the antigen specific cells)

The activation of lymphocytes by a polyclonal activator can help investigate abnormal lymphocyte functions

MEASURING LYMPHOCYTE ACTIVITY

For detection of immunodeficiencies affecting T and/or B cell functions

POLYCLONAL ACTIVATION OF B AND T CELLS

Lectins (like concavalin A and PHA) act through crosslinking receptors

Intracellular signaling cascade activators (PMA – PKC activator, Ionomycin – increased intracellular Ca2+ levels)

Specific antibodies (anti-IgM, anti-CD3, anti-TCR)

POLYCLONAL B CELL ACTIVATORS

POLYCLONAL T CELL ACTIVATORS

Pokeweed mitogen (PWM)

Staphylococcus protein A superantigen (SpA)

Epstein Bar Virus (EBV) (transforming)

Anti-IgM antibody

Phytohaemagglutinin (PHA)

Concanavalin A (ConA)

Anti-CD3, Anti-TCR antibodies

Canavalia ensiform

isP

hytolacca ame

ricana P

haseolus vulgaris

Receptor crosslinking(immediate)

phosphorylation steps(seconds-minutes)

- Western blot

i.c. Ca2+ increase - flow cytometry- fluorescent microscopy

Gene activation - qRT-PCR mRNA- Western blot protein

Cytokine synthesis

Cytokine secretion

- i.c. cytometry

- ELISA- ELISPOT

Antigen receptors (TCR, BCR), cytokine receptors, etc.

Viability/apoptosis - dies specific to dead cells

Cell division - 3H-thymidine- CFSE- MTT

Lymphocyte activationThe examination often requires

specific Ag-Ab reactions

Flu

ore

scen

ce p

rop

ort

ion

al

wit

h I

ntr

acel

lula

r C

a2+ l

evel

time

basic signal

activation of cells

Fluo-3or

Indo-1

MEASUREMENT OF CA2+ SIGNAL BY FLOW CYTOMETRY

An increase in cytoplasmic Ca2+ levels can be detected by fluorescent indicator dyes

/Fluo-3 or Indo-1/

INTRACELLULAR CYTOKINE DETECTION BY IMMUNOFLUORESCENCE

cytokine specific antibodies with fluorescent labeling

cytokines

the cell membrane should be permeabilized (detergent)

but first the cells should be fixed to avoid decomposition (using e.g. aldehyde fixation)

optionally the cells can also be labeled by cell type (CD marker) specific antibodies

Activation of cells can be monitored by the detection of mRNA transcription of the activated genes

e.g. activation of cytokine genes

QUANTITATIVE (REAL-TIME) PCR (qPCR/qRT-PCR)

cells RNA isolation RNA reverse transcription (RT-PCR) cDNA cDNA polymerase chain reaction (PCR)

determination of quantity

(investigation of gene activation on protein level WB)

INVESTIGATION OF GENE ACTIVATION

the more mRNA the sample contains, the less time (cycles) it will take to reach the threshold

ELISPOTEnzyme Linked Immuno-Spot

Similar principles as in ELISA

Determination of the number of cells that produce Ig, cytokines, chemokines, granzymes and other soluble effector molecules

Sensitive. Allows the determination of 1 activated cell among 300,000 others. (Can reveal activated effector cells not only after polyclonal but after antigen specific activation).

- coating with antigen specific capture antibodies

- blocking

- administration of the cells

- administration of biotin conjugated antigen-specific secondary antibody

- avidin-enzyme conjugate

- administration of the insoluble chromogenic substrate (AEC 3-amino-9-ethylcarbazol)

(activation, incubation)

- washing

A spot showing the place of the

cytokine producing cell

Upper view of a well on an ELISPOT plate with the generated spots

ELISPOTEnzyme Linked Immuno-Spot

ELISPOTEnzyme Linked Immuno-Spot

Spot number and size determination is valuated slowly and manually by microscopy or using “ELISPOT plate reader” which is fast and standardizable

VIABILITY ASSAYS

MTT (Dimethyl thiazolyl diphenyl tetrazolium salt)Colorimetric test for measuring viability (apoptotic cells). NADPH-dependent cellular oxyreductase enzymes that reduce MTT dye to an insoluble purple color (formazan).

PI (propidium iodide)A fluorescent molecule intercalating with nucleic acids for measuring cell viability by flow cytometry. It is impermeable to viable cells.

7-AAD (7-aminoactinomycin)A fluorescent chemical intercalating with dsDNA. Won’t pass intact cells so is used for cell viability by flow cytometry.

PROLIFERATION ASSAYS

3H-labeled thymidine- measures the increasing DNA content by β decomposition, and does not answer the numbers of cell division, and the dividing cell number.

Bromodeoxyuridin (BrdU) A Thymidine-analogue can be administered to experimental animals, or cell cultures, and the proliferating cells can be detected by labelling with BrdU specific antibody (microscopy, FACS).

CFSE (Carboxyfluorescein succinimidyl ester)A fluorescent dye easily penetrating cells binding intracellular amine structures for long periods. Studies of cell divisions, prolifearation, migration and positioning.

CFSETRACKING THE CELL DIVISIONS

„Cell tracer” dye enters the cell, and becomes trapped there

The apolar CFSE can bind covalently to the cellular proteins

Progressively halved within daughter cells

Used in vitro and in vivo to monitor lymphocyte proliferation

CFSE-labeled cells that were not treated with polyclonal activator

(control)