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