FlowCellect Kits For Quantification Of Chemokine ... - EMD Millipore

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Introduction. The single cell analysis capabilities inherent in flow cytometry ... providing highly accurate and robust data sets on low volume, small ... In addition, the instrument software ... EasyCyte™ Plus bench top flow cytometer, which uses.
™ FlowCellect

Kits For Quantification Of Chemokine Receptor Expression Using Guava EasyCyte™ Plus

Mark Santos1, Wenying Zhang1, Joelle Wolfson1, James Navarro1, Patrick Schneider1, Jason Whalley1 and Matthew Hsu1 § Millipore Corporation, 28820 Single Oak Drive, Temecula, CA92590;

Abstract

4: Calculation of the receptor numbers (per cell) based on the FlowCellect™ assay results:

3: Kit Validation for GPCR identification and quantification: Can the antibody detect endogenous levels of GPCR expression on a native cell system? Moreover, is quantifying receptor numbers feasible using this method?

Automated Data Acquisition and Analysis Software

As described by Schertler et al. (1992), a rough guide for converting expression levels has been created for GPCRs relative to the maximal number of binding sites (Bmax) determined by radioligand binding assay. For a given cell line, approximately 106 molecules per cell correspond to approximately 10 pmol/mg of membrane protein.

CXCR4 Expression on Jurkat T Cells Plot3: Gated by : Gate 1

200

Optimally Formulated Multi-color Kits for Flow

6% MFI=8.54

Wild Type “negative” control cells

98% MFI=588

150

(< 20,000 receptors/cell; MFI=8.54) CXCR4/Chem-1 “positive” control cells

96% MFI=273

(380,000 receptors/cell; MFI=588)

50

Count 100

• Determined by radioligand binding assay, ChemiScreen™ CXCR4 membranes yield a Bmax value of 3.8 pmol/mg. This corresponds to approx. 380,000 receptors per cell. • In flow analysis, using the Guava EasyCyte™ Plus system, a mean fluorescence intensity (MFI) of 588 is determined for the positive control cells. • A native cell line (Jurkat T cells) with CXCR4 endogenous expression produces a MFI of 273 when evaluated on the same system.

Jurkat T cells (Native cell line)

M1

0

(MFI=273, equivalent to approx. 176,320 receptors/cell)

10e0

10e1 10e2 10e3 Yellow Fluorescence (YLW-HLog)

10e4

Total # of cell = (native cell MFI / “-” control MFI) surface receptors (“+” control MFI / “-” control MFI)

Figure 2. The histograms above illustrate how using Millipore’s FlowCellect™ GPCR Surface Identification Kit can be used to quantitatively measure cell surface expression for any given cell type. The histogram on the far right (pink) 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) is the negative control cells.

Micro-Volume Bench Top Instruments

Assay Overview

Values can be further extrapolated to 1.76 pmol/mg protein for the native (Jurkat T) cell line

6: Summary of Results Comparing Flow vs. Radioligand Binding: Provided with the calculation for converting pmol/mg values to number of receptors per cell as noted in Schertler et al., can we compare values obtained using flow cytometry to those gathered from Radioligand Binding Assay?

Provided with a fully characterized positive control cell line, do the results gathered from a saturation binding assay on a native cell line correlate to values obtained utilizing flow as the application?

A. CXCR4/Chem-1 (recombinant)

Fix cells for 20 minutes, followed by three wash steps to remove any residual fixation solution from cell cultures.

↓ Incubate with Primary (1º) antibody for one hour, followed by three wash steps to remove any residual antibody from cell suspension.

↓ Incubate with the Secondary (2º) antibody for 45 minutes to one hour, followed by three wash steps to remove any residual antibody from cell suspension.

90000 80000 70000 60000 50000 40000 30000 20000 10000 0 0.0

B. Jurkat T Cells (native expression) 14000

NSB TB SB

0.5

1.0

[

125

1.5

2.0

2.5

I] - SDF1-α α (nM)

Saturation binding using CXCR4 stable cell line



[125I]-SDF-1α bound (cpm)



TB NSB SB

12000 10000 8000 6000 4000 2000 0 0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

[125I]-SDF-1α α (nM)

Saturation binding using native T cells

Begin cellular analysis by acquiring cell samples. Figure 3. Saturation Radioligand Binding Curves. Using a radioligand binding assay, we compared the values obtained from Millipore’s ChemiScreen™ CXCR4 stable cell line membrane preparation (Catalog #: HTS004M) (A) versus those of a native CXCR4 expressing cell line, Jurkat T cells (B). In both cases, Bmax values were determined and compared (Table 1).

↓ Collect data and analyze cellular distribution.

Summary

Result 1: Evaluation of Antibody for Flow Applications:

2: Validation of Frozen Control Cells for Receptor

Does the antibody clearly identify GPCR expression with a great degree of confidence relative to the isotype control?

Expression Level: Does the antibody clearly differentiate between the frozen negative “null” control cells and the frozen positive control cells?

A. CXCR2 with IgG2a Isotype Control

B. CCR7 positive control vs. WT 150

97.7% MFI=533

1.9% MFI=5.3

99.4% MFI=1286

50

Count 100

Count 75

150

2.3% MFI=3.2

Plot3: Gated by : Gate 1

113

200

Plot3: Gated by : Gate 1

M1

0

0

M1

10e0

10e1 10e2 10e3 C XC R 2-F ITC (GR N -H L o g )

10e4

10e0

10e1 10e2 10e3 Yello w F lu o r escen ce (YL W -H L o g )

10e4

Figure 1. Antibody and Frozen Control Cell Evaluation. The histograms above illustrate the following: The provided antibody has been optimized and can detect the GPCR of interest relative to the isotype control (A); and to further confirm antibody specificity and validation of the provided control ChemiScreen™ frozen cell cultures (B).

Millipore, Upstate, Chemicon, and Linco are registered trademarks of Millipore Corporation. The M mark is a trademark of Millipore Corporation. Guava, Guava Technologies, the Guava logo, and all other trademarks are the property of Guava Technologies, Inc. Lit. No. PS2521EN00 Printed in U.S.A. 09/08 © 2008 Millipore Corporation, Billerica, MA 01821 U.S.A. All rights reserved

*As calculated: (273 / 8.54) =0.464 or 46.4% (588 / 8.54)

Where 46.4% of 380,000 receptors per cell equals approximately 176,320 receptors per native cell

5: Verification of Flow Results by Radioligand Binding Assays:

Collect cells in culture by mild enzyme treatment (using Accutase or Cell dissociation buffer). For “stickier” cells, trypsin treatment may be preferred. Determine viability and perform cell count.

Introduction

A case study demonstrating cell surface receptor quantification and identification on a native cell line is presented using CXCR4 and Jurkat T cells as a model. Jurkat cells are an immortalized line of T lymphocyte cells that are used to study acute T cell leukemia and T cell signaling. CXCR4 has been indicated to be expressed on T cells and serves as an HIV co-receptor. In HIV-positive individuals, the level of expression of CXCR4 is down-regulated in T cells as a result of receptor internalization. By using Millipore’s FlowCellect™ GPCR identification and quantification kit, we have been able to confirm the expression level of CXCR4 on T cells. We were able to quantitatively measure cell surface expression levels accurately when compared to radioligand binding using flow as the primary method for detection.

How to identify and quantify the receptor level on your cells with FlowCellect™ kits?

Millipore and Guava Technologies Provide Integrated Cell Analysis Solutions

FlowCellect™ kits have been optimized and validated on a Guava EasyCyte™ Plus bench top flow cytometer, which uses microcapillary “flow cell” technology that enables the analysis of small sample volumes without using sheath fluid.

The single cell analysis capabilities inherent in flow cytometry facilitates detection on thousands of cells per second providing robust, high content, information on a given population of cells. The Guava EasyCyte™ Plus flow cytometer adds the key advantage of providing highly accurate and robust data sets on low volume, small sample size cell populations. In addition, the instrument software provides an intuitive user interface and offers data acquisition and analysis capabilities. We have evaluated the feasibility of identifying and quantitatively measuring GPCR receptor expression using this system. Based on our results, we have determined that using the Guava EasyCyte™ Plus flow cytometer in combination with the FlowCellect™ GPCR assay kits has proven to be a useful tool for any researcher interested in cell line characterization and assay development.

Corresponding Author: [email protected]

[125I] - SDF1-α Bound (cpm)

Chemokine receptors are very attractive drug targets because they regulate leukocyte activation and trafficking. Many of these targets have been indicated to play a key role in certain diseases such as HIV infection, Rheumatoid Arthritis, and Diabetes. The expression levels of these receptors often reflect the different states of autoimmune disorders and inflammation. Chemokine receptors are members of G-Protein Coupled Receptors (GPCRs) family. They include ten CC chemokine receptors, seven CXC chemokine receptors, and a CX3C chemokine receptor. In order to allow for rapid identification and quantification of the receptor expression levels on the leukocyte surface, we have developed 12 FlowCellect™ GPCR surface quantification kits based on flow cytometry validated antibodies and pharmacologically characterized control cell lines for reference of receptor expression level. The antibody against each receptor has been validated for flow cytometry applications using Guava instruments, and optimized to detect a wide range of receptor expression levels, ranging from endogenous receptors to overexpressing cell lines. The reference positive control cells are made from Millipore’s proprietary ChemiScreen™ GPCR stable cell lines, preserved and frozen. The null host cell line is used as negative control. These control cell lines have well characterized receptor expression levels, determined by using pharmacological techniques such as radioligand saturation binding assays (measuring Bmax value) for the purpose of quantification. By using Millipore’s GPCR surface identification and quantification kit, the researcher now has the ability to identify a native GPCR expressing cell for biological assay development and estimate the receptor expression level without the need to resort to extremely laborious and costly binding assays. Moreover, the researcher can now easily select clonal stable cell lines for these GPCRs with greater confidence. Lastly, the researcher now has the ability to monitor the changes of these Gprotein coupled receptor expression levels during a given disease state.

§

38

1

Test Model

CXCR4/Chem-1 (Cat#: HTS004M)

Jurkat T Cell (Endogenous Expression)

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 (determined from radioligand binding assay)

3.8 pmol/mg

1.5 pmol/mg

RECEPTORS PER CELL (*Derived from binding assay)

380,000 per cell

150,000 per cell

RECEPTORS PER CELL (*Derived from flow cytometry)

----------

~ 176,320 per cell

Table 1. Comparison of Positive control cells versus a Native cell line. The table above summarizes values obtained when performing traditional radioligand binding assays to flow cytometry in determining Bmax values or receptors per cell. Resulting values indicate that the predicted cell surface receptors equate to the values generated when performing flow cytometry methods.

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References

• We have demonstrated that using Millipore’s FlowCellect™ Chemokine Receptor Surface Expression ID kit is an effective tool in identifying cell surface expression on any given cell type. • Provided in each kit are Millipore’s patented ChemiScreen™ GPCR cell lines (fixed) which have been fully characterized using Pharmacology techniques, which can serve as excellent controls to quantify receptor numbers on the cell surface.

•The Guava EasyCyte™ Plus system utilizes patented flow cell micro-capillary technology which allows very small sample volumes during cellular analysis.

1. Schertler GFX (1992). Curr Opin Struct Biol. 2: 5-34-544. 2. J. Di Salvo et al. (2000). European Journal of Pharmacology. 409: 143-154. 3. Hesselgesser, J. et al. (1998). J. Immunol. 160: 877-883.