Cellular Assays of Chemokine Receptor Activation

Amanda E.I. Proudfoot1, Christine A. Power1, Dennis J. Church1, Dulce Soler2, Matthias Mack3

1 Serono Pharmaceutical Research Institute, Plan‐les‐Ouates, Switzerland, 2 Millennium Pharmaceuticals, Cambridge, Massachusetts, 3 Ludwig‐Maximilians‐University of Munich, Munich, Germany
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 12.4
DOI:  10.1002/0471141755.ph1204s14
Online Posting Date:  November, 2001
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Abstract

This unit describes procedures for measuring the activation of chemokine receptors by their ligands. Chemokines are chemoattractant proteins and two assays are described for analyzing the chemoattractant properties (chemotaxis) of these proteins. Chemokine receptors are coupled to G proteins, and activation of the receptors results in the mobilization of intracellular Ca2+ stores. Two assays for the measurement of this activity are described which employ calcium‐sensitive dyes, one for adherent and one for non‐adherent cells. In addition, an assay for quantitating chemokine receptor downmodulation and subsequent recycling events is described. The assays are applicable to both purified leukocytes and stably expressed recombinant chemokine receptors.This unit describes procedures for measuring the activation of chemokine receptors by their ligands.

     
 
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Table of Contents

  • Basic Protocol 1: Modified Micro‐Boyden Chamber Chemotaxis Assay
  • Alternate Protocol 1: 96‐Well Format Chemotaxis Assay
  • Alternate Protocol 2: 24‐Well Transwell Chemotaxis Assay
  • Alternate Protocol 3: Transendothelial Chemotaxis Assay
  • Basic Protocol 2: Assay of Chemokine Receptor‐Induced Increases in Cytosolic Free Calcium ([Ca2+]i) in Nonadherent Cells
  • Alternate Protocol 4: High‐Throughput Measurement of [Ca2+]i in Adherent Cells
  • Basic Protocol 3: Chemokine Receptor Down‐Modulation Assay
  • Basic Protocol 4: Analysis of Receptor Recycling After Down‐Modulation
  • Support Protocol 1: Leukocyte Purification
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Modified Micro‐Boyden Chamber Chemotaxis Assay

  Materials
  • Chemokine (e.g., PeproTech, R&D Systems, Dictagene, Gryphon Sciences)
  • Boyden chemotaxis medium (see recipe)
  • Test compound (optional)
  • Purified leukocyte (see protocol 9) or transfected cell line suspension
  • 1× phosphate buffered saline (PBS; see recipe)
  • 100% methanol
  • 12.5% (w/v) Field's stain B (red)
  • 12.5% (w/v) Field's stain A (blue)
  • 48‐well microchemotaxis chamber (Neuro Probe)
  • 3, 5, 8, or 10‐µm polyvinylpyrolidone‐free (PVDF) membranes (Corning; Table 12.4.1)
  • 48‐well accessory pack (Neuro Probe): clamps, clips, and rubber wiper blade
  • 76 × 26–mm glass microscope slides (Merck)
  • Marker
  • Absorbent paper
  • IBas image analyzer (Kontron) or light microscope (e.g., Axioskop model D‐7082; Carl Zeiss) equipped with a video camera (AVT‐Horn)
    Table 2.4.1   MaterialsMicro‐Boyden Chamber Assay Parameters for Various Cell Types

    Cell type Filter pore size (µm) Cell concentrations (cells/ml) Incubation time (min)
    Lymphocytes 8 2 × 106 90–120
    Monocytes 5 2.5 × 106 30–45
    Neutrophils 3 1 × 106 30
    RBL transfectants 10 2 × 106 30–45

Alternate Protocol 1: 96‐Well Format Chemotaxis Assay

  Materials
  • Purified leukocytes (see protocol 9)
  • RPMI medium without phenol red (Life Technologies) containing 5% (w/v) heat‐inactivated FBS ( appendix 2A)
  • Chemokines (PeproTech)—e.g., IFN‐inducible T‐cell α chemoattractant (ITAC), IFN‐inducible protein 10 (IP‐10), monokine induced by IFN‐γ (MIG)
  • 1× PBS (see recipe)
  • Cyquant GR solution (Molecular Probes)
  • ChemoTX microplate with filter plate of 5‐µm pore size (Neuroprobe)
  • Funnel plate (Neuroprobe)
  • 96‐well black‐walled plate (Porvair)
  • Centrifuge with microplate carrier
  • Fluorescence plate reader (e.g., Wallac Victor2)

Alternate Protocol 2: 24‐Well Transwell Chemotaxis Assay

  Materials
  • 0.5 × 106 purified leukocytes/ml (see protocol 9) or transfected cells in appropriate medium
  • 0.5 M butyric acid (optional)
  • Chemotaxis medium—e.g., RPMI 1640 (Life Technologies)/0.5% (w/v) BSA (transfectants)
  • Chemokines
  • 24‐well transwell chemotaxis plates (e.g., Costar Corning) with polycarbonate membranes of 6.5 mm diameter and a pore size of either 3—i.e., for neutrophils, monocytes, lymphocytes, and L1.2 transfectants—or 8 µm—i.e., for lymphocytes, and rat basophilic leukemic‐1 (RBL‐1) cell transfectants
  • Fluorescence‐activated cell sorter (i.e., FACSCalibur; Becton Dickinson) and FACS tubes

Alternate Protocol 3: Transendothelial Chemotaxis Assay

  • ECV 304 tissue culture media (see recipe)
  • Endothelial cell line ECV 304 cells (ECACC #92091712; http://www.ecacc.org)
  • Transendothelial chemotaxis medium (see recipe)

Basic Protocol 2: Assay of Chemokine Receptor‐Induced Increases in Cytosolic Free Calcium ([Ca2+]i) in Nonadherent Cells

  Materials
  • Chemokine: purified chemokine, supernatant from a cell line expressing a recombinant chemokine of interest, or fractions containing a chemokine purified from tissue
  • Krebs‐Ringer buffer, fresh (see recipe)
  • Cell lines expressing the chemokine receptor of interest (recombinant CHOs, HEKs, L1.2s, RBLs), or purified leukocytes (see protocol 9)
  • Appropriate culture medium
  • 1× PBS (see recipe) with and without 1 mM EDTA ( appendix 2A)
  • 0.18 M EGTA/1.40 M Tris⋅Cl ( appendix 2A)/5% (v/v) digitonin
  • 1 M HCl/0.5 M CaCl 2
  • 175‐cm2 culture flasks
  • Hemacytometer
  • Aluminum foil
  • 1400‐µl, 10 × 4–mm path lengths (320‐nm cutoff) fluorescence cuvettes (HELLMA) and 6 × 3 × 3–mm magnetic stir bars
  • Spectrofluorometer (e.g., Jasco FP‐777)

Alternate Protocol 4: High‐Throughput Measurement of [Ca2+]i in Adherent Cells

  Materials
  • Adherent cells expressing the chemokine receptor of interest
  • Appropriate culture medium
  • Purified chemokine, supernatant from a cell line expressing the recombinant chemokine of interest, or fractions containing a chemokine purified from tissue
  • Fluorometric imaging plate reader (FLIPR) assay buffer (see recipe)
  • Serum‐free Dulbecco's modified Eagle medium (DMEM)‐F12 containing 2 mM glutamine and 100 U/ml penicillin‐streptomycin (Life Technologies)
  • Serum‐free DMEM‐F12 containing 1 µM cyclosporin A (Calbiochem), 1 µM probenicid (Sigma), and 4 µM fluo‐(2, 3, or 4)‐AM (previously dissolved in 20% pluronic acid from a 1 mg/ml stock; Molecular Probes)
  • 96‐well, black‐walled, clear‐bottomed, cell culture plates (Polyfiltronics)
  • 96‐well conical‐bottomed assay plates (Falcon or Costar)
  • Multichannel pipettor
  • Fluorometric imaging plate reader (FLIPR; Molecular Devices; also see unit 9.2)

Basic Protocol 3: Chemokine Receptor Down‐Modulation Assay

  Materials
  • Chemokines to be tested
  • Appropriate culture medium
  • 4 × 106 cells/ml cell lines or primary cells expressing chemokine receptors, 4° and 37°C
  • Appropriate antichemokine receptor mono‐ or polyclonal antibodies, 4°C
  • Isotype control antibody
  • Two 96‐well U‐bottom plates
  • Microplate shaker
  • Centrifuge with microplate carrier
  • Fluorescence‐activated cell sorter (i.e., FACSCalibur; Becton Dickinson) and FACS tubes

Basic Protocol 4: Analysis of Receptor Recycling After Down‐Modulation

  Materials
  • Human blood, buffy coats, or plasma, freshly drawn
  • Anticoagulant solution: 1 vol 0.1 M EDTA ( appendix 2A) and 2 vol 3% (w/v) dextran/3% (w/v) glucose in water
  • 1× PBS (see recipe)
  • Ficoll (Amersham Pharmacia Biotech)
  • RPMI 1640 medium (Life Technologies) containing 5% heat‐inactivated FBS ( appendix 2A)/2 mM glutamine/25 mM HEPES, pH 7.2
  • 0.4% (w/v) trypan blue
  • 1 × 106 sheep red blood cells/ml (RBCs; BioMérieux or equivalent)
  • 0.2% and 1.8% NaCl solutions
  • HEPES‐buffered Hank's balanced salt solution (HBSS; Life Technologies or equivalent), pH 7.4 containing 1 mg/ml BSA
  • M‐450 Dynabeads (Dynal)
  • Anti‐CD16 monoclonal antibody (e.g., clone 3G8; Dutch Red Cross)
  • 15‐ and 50‐ml conical tubes, sterile
  • Neubauer chamber (Merck)
  • Hemacytometer (Merck)
  • Fluorescence‐activated cell sorter (i.e., FACSCalibur; Becton Dickinson)
  • 0.45‐µm syringe filter, sterile (Millipore)
  • Rotary shaker
  • T cell–enrichment column (e.g., Total T Cell Enrichment Column; R&D Systems)
  • Magnet
CAUTION: Human tissue is a biohazard and should be handled according to the Occupational Safety and Health Administration (OSHA) regulations for blood‐borne pathogens (29CFR1910‐1030). This document is available at http://www.osha-slc.gov/OshStd_data/1910_1030.html Institutional guidelines must be strictly followed.
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Figures

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Literature Cited

Literature Cited
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