flowcellect™ chemokine receptor surfaceexpression quantification kits
DESCRIPTION
EMD Millipore offers a new range of FlowCellect kits that are specifically developed and validated for flow cytometry applications. The FlowCellect Chemokine Receptor Surface Expression Quantification Kits are designed to provide rapid, sensitive assessments in many different cell types, creating a new standard in chemokine receptor quantification.Flow cytometry is a powerful tool for chemokine receptor research. By delivering robust, high content information on a cell-by-cell basis, flow cytometry provides high quality, reproducible data in far less time than traditional methods. FlowCellect kits use fluorescence detection, coupled with positive and negative control cells, to avoid the hazard and expense of radioligand binding assays. Plus, all FlowCellect kits are optimized on Guava® flow cytometry systems, so you can save valuable time when you use the kits and systems together.Chemokines play critical roles in a number of cellular processes. They are involved in leukocyte trafficking and activation, mediated by 7-TM chemokine receptors. They are also widely expressed on immune system cells, and can be important indicators of autoimmune disorders and HIV infections. Rapid assessments of chemokine receptor expression can thus be used in a variety of applications, including infectious disease and inflammation research. This crucial research is now made easier with the sensitive, validated FlowCellect Chemokine Receptor Surface Expression Quantification kits.TRANSCRIPT
Data SheetData Sheet
FeaturesAssay:
• Quantitativeidentificationofchemokinereceptor
surfaceexpression
• Includespharmacologicallycharacterizedpositive
andnegativecontrolcells
• Optimizedantibodiesarevalidatedforflow
cytometry
• Containseverythingyouneedtorun100samples
• Sameaccuracyasradioactiveassays,without
thedanger
Samples:
• Designedforhumancells
Compatibility:
• OptimizedforGuavaEasyCyte™
Millipore offers a new range of FlowCellect kits that are
specifically developed and validated for flow cytometry
applications. The FlowCellect Chemokine Receptor
Surface Expression Quantification Kits are designed to
provide rapid, sensitive assessments in many different
cell types, creating a new standard in chemokine
receptor quantification.
Flow cytometry is a powerful tool for chemokine
receptor research. By delivering robust, high content
information on a cell-by-cell basis, flow cytometry
provides high quality, reproducible data in far less
time than traditional methods. FlowCellect kits use
fluorescence detection, coupled with positive and
negative control cells, to avoid the hazard and expense
of radioligand binding assays. Plus, all FlowCellect kits
are optimized on Guava® flow cytometry systems, so
you can save valuable time when you use the kits and
systems together.
Chemokines play critical roles in a number of
cellular processes. They are involved in leukocyte
trafficking and activation, mediated by 7-TM chemokine
receptors. They are also widely expressed on immune
system cells, and can be important indicators of
autoimmune disorders and HIV infections. Rapid
assessments of chemokine receptor expression can
thus be used in a variety of applications, including
infectious disease and inflammation research. This
crucial research is now made easier with the sensitive,
validated FlowCellect Chemokine Receptor Surface
Expression Quantification kits.
FlowCellect™ChemokineReceptorSurfaceExpressionQuantificationKits
2
The CXCR2 antibody (blue) labels chemokine receptors with
a great degree of confidence relative to the isotype control
(red) (A). Likewise, the CCR7 antibody easily differentiates
ADvAntAgeS•Optimized:Reagents and protocols have already been
optimized to ensure that you get the best results on every
sample, every time.
•Quickandeasy:FlowCellect kits are designed for straight-
forward detection and minimal hands-on time.
How it workS
ChemokineReceptor
CalciumChannel
Chemokines
JAK2
PKCCa2+
PYK2
GDP
GTP
Gα
p38 JNKPDK
Akt/PKB
Akt/PKB Pathway
Rho Pathway
Ras Pathway
CrkCAS
PLC-γ
PI3K
STAT5
Gα
Gβ Gγ
Gβ Gγ
PLCβ
PIP2
IP3DAG
PKC-β
GRK
β-Arrestin
PTK Rho
MAPKs
PLA2 Ca2+
NFκB
c-Jun
c-Fos Elk1
SRF
InternalizationDegradation
Recycling Cell Activation
Gene Expressionand Apoptosis
GRB2
Ras
Ca2+
In addition to antibodies, buffers, and fixatives, FlowCellect
Chemokine Receptor Surface Expression Quantification kits
include positive and negative control cells to help calculate
and confirm your results. These cells are frozen samples of
Millipore’s ChemiScreen™ myeloid cell lines; wild-type cells serve
validated Antibodies
FlowCellect antibodies clearly identify chemokine expression in comparison to controls.
A.CXCR2withlgG2aIsotypeControl B. CCR7PositiveControlvs.WT
as the negative control, while cells from the clonal stable cell
line that over-expresses the relevant chemokine receptor are
used as the positive control. All cell lines have been fully char-
acterized using pharmacology techniques, such as radioligand
binding, to give you complete confidence in your data.
•Non-radioactive:You can accurately quantify receptor
levels without the hazards of radioligand binding assays.
•Smallsamples:Guava’s patented microcapillary flow
cytometers give you more data from smaller samples,
so you can conserve your samples.
between the negative (wild-type, red) and positive
(overexpressing, blue) ChemiScreen control cells that are
included in the kit (B).
3
The histogram on the far right (blue)
represents the positive control
cells, the histogram in the middle
(green) represents the native cell line
(Jurkat T cells), and the histogram
on the far left (red) represents the
negative control cells.
TestModel CXCR4/Chem-1 JurkatTCell
Cell Type Recombinant Native
Mode Control Test Sample
MFI Value 588 273
Change in MFI relative to the negative control 68.9 32.0
Percentage (%) change relative to positive control ---------- 46.4%
Bmax value (radioligand binding assay) 3.8 pmol/mg 1.5 pmol/mg
Receptors per cell (radioligand binding assay) 380,000 150,000
Receptors per cell (flow cytometry) ---------- ~ 176,320
Chemokine receptor Quantification
FlowCellect and radioligand Binding Comparison
FlowCellect kits detect and quantify endogenous chemokine receptor expression on native cells.
Results from the FlowCellect kits correlate with saturation binding assays, demonstrating the accuracy of these kits.
CXCR4ExpressiononJurkatTCells
A.CXCR4/Chem-1(recombinant)
α
α
B.JurkatTCells(nativeexpression)
α
α
The FlowCellect Chemokine Receptor Surface Expression
Identification and Quantification Kits can be used to calculate
chemokine receptor expression levels according to the
following equation:
Total#ofcell = (nativecellMFI/-controlMFI)
surfacereceptors (+controlMFI/-controlMFI)
In flow analysis, using the Guava EasyCyte Plus system, a mean
fluorescence intensity (MFI) of 588 is determined for the positive
control cells, and a native cell line (Jurkat T cells) with CXCR4
endogenous expression produces a MFI of 273. So, we have:
(273/8.54)
(588/8.54)=0.464or46.4%
Where 46.4% of 380,000 receptors per cell = approximately
176,320 receptors per native cell.
Values can be further extrapolated to 1.76 pmol/mg protein
for the native (Jurkat T) cell line.
Calculating Chemokine expression Levels
Values from CXCR4 radioligand binding assays on a ChemiScreen CXCR4-positive cell line (A) and Jurkat T cells (B) were compared
to results from a FlowCellect flow cytometry assay.
www.millipore.com www.guavatechnologies.com
orDering inFormAtionFlowCellect kits Catalogue no.
FlowCellect Chemokine Receptor CCR1 Surface Expression Quantification Kit FCCR100410
FlowCellect Chemokine Receptor CCR2B Surface Expression Quantification Kit FCCR200411
FlowCellect Chemokine Receptor CCR3 Surface Expression Quantification Kit FCCR300412
FlowCellect Chemokine Receptor CCR4 Surface Expression Quantification Kit FCCR400413
FlowCellect Chemokine Receptor CCR6 Surface Expression Quantification Kit FCCR600414
FlowCellect Chemokine Receptor CCR7 Surface Expression Quantification Kit FCCR700415
FlowCellect Chemokine Receptor CXCR1 Surface Expression Quantification Kit FCXR100420
FlowCellect Chemokine Receptor CXCR2 Surface Expression Quantification Kit FCXR200421
FlowCellect Chemokine Receptor CXCR3 Surface Expression Quantification Kit FCXR300422
FlowCellect Chemokine Receptor CXCR4 Surface Expression Quantification Kit FCXR400423
FlowCellect Chemokine Receptor CXCR6 Surface Expression Quantification Kit FCXR600424
Millipore is a registered trademark of Millipore Corporation. The M mark, ChemiScreen, and FlowCellect are trademarks of Millipore Corporation. Guava and Guava Technologies are registered trademarks of Guava Technologies, Inc. EasyCyte is a trademark of Guava Technologies, Inc.Lit. No. DS1189EN00 10/08 Printed in U.S.A. BS-GEN-08-01066 © 2008 Millipore Corporation, Billerica, MA 01821 U.S.A. All rights reserved.
Audience
Flow Cytometry Systems
FlowCellect kits
guavaAssays & kits Service
Europe&NorthAmerica
Academic&Government n n n n
Pharma&Biotech l n l n
AllOtherCountries n n n n
n Millipore Corporation l Guava Technologies
To learn more about our integrated
solutions for flow cytometry, please
contact Millipore or Guava Technologies.
The chart (right) shows which company
serves your organization.
To stay up-to-date on our joint product
and service offerings, please visit
www.millipore.com/flowcytometry.
ChemiScreen Cell Lines Catalogue no.
Human Recombinant CCR1 Cell Line HTS005C
Human Recombinant CCR2B Cell Line HTS007C
Human Recombinant CCR3 Cell Line HTS008C
Human Recombinant CCR4 Cell Line HTS009C
Human Recombinant CCR6 Cell Line HTS011C
Human Recombinant CCR7 Cell Line HTS012C
ChemiScreen Cell Lines Catalogue no.
Human Recombinant CXCR1 Cell Line HTS001C
Human Recombinant CXCR2 Cell Line HTS002C
Human Recombinant CXCR3 Cell Line HTS003C
Human Recombinant CXCR4 Cell Line HTS004C
Human Recombinant CXCR6 Cell Line HTS054C
Human Recombinant Chem-1 WT Cell Line HTSCHEM1