Detection of Cytokine Receptors by Flow Cytometry

Heddy Zola1

1 Child Health Research Institute, Adelaide, Australia
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 6.21
DOI:  10.1002/0471142735.im0621s26
Online Posting Date:  May, 2001
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Abstract

This unit describes three protocols for detecting cytokine receptors on human peripheral blood mononuclear cells or murine splenic lymphocytes via immunofluorescence labeling. The three protocols have in common the use of phycoerythrin as a highly effective fluorescent dye, multiple‐layer staining to increase sensitivity, and selection of reagents for maximum sensitivity. The first procedure uses monoclonal antibody (MAb) against receptor protein, detected with biotinylated anti‐Ig (directed against the species in which the MAb is made), which in turn is stained with phycoerythrin‐streptavidin (PE‐SA). The second procedure uses biotinylated cytokines in the first step, followed by PE‐SA. Finally, a third procedure describes multiparameter analysis involving second and third fluorophores introduced as direct antibody conjugates, which is used to study subset expression of receptors. Instructions are also provided for titrating MAbs and cytokines, as well as how to evaluate detection reagents such as anti‐Ig conjugates and fluorophores.

     
 
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Table of Contents

  • Basic Protocol 1: Detection of Human and Murine Cytokine Receptors with Monoclonal Anti‐Receptor Antibodies
  • Alternate Protocol 1: Detection of Cytokine Receptors with Labeled Cytokines
  • Alternate Protocol 2: Use of Multiparameter Analysis to Detect Cytokine Receptor Expression by Lymphocyte Subpopulations
  • Support Protocol 1: Titration of Monoclonal Antibodies and Cytokines
  • Support Protocol 2: Evaluation of Detection Reagents
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Detection of Human and Murine Cytokine Receptors with Monoclonal Anti‐Receptor Antibodies

  Materials
  • Heparinized human peripheral blood samples ( appendix 3F)
  • recipePBS/azide (see recipe), 4°C
  • MAb against human cytokine receptor (Table 6.21.1), concentration determined by titration (see protocol 4)
    Table 6.1.1   MaterialsMonoclonal Antibodies Against Human and Murine Cytokine Receptors

    Cytokine receptor a CD antigen b Supplier c
    Human Murine
    IL‐1R type 1 (p80) CD121a GN, PG GN, PG
    IL‐1R type 2 CD121b GN PG
    IL‐2Rα CD25 Many Many
    IL‐2Rβ CD122 BD, CI, PG EN, PG
    IL‐2Rγ (common chain) CD132 PG PG
    IL‐3Rα CDw123 PG, SA PG
    IL‐3R/IL‐5R/GM‐CSF common receptor CDw131 PG, SA
    IL‐4R CD124 GN, CI GN
    IL‐5R CDw125 PG
    IL‐6R CD126 BS, ST, CI GN, PG
    gp130 CD130 ST, PG
    IL‐7R CD127 GN, CI
    IL‐8R CDw128 PG
    TNFR p55 CD120a GN
    TNFR p75 CD120b GN
    IFN‐γR CDw119 GN, PG PG
    GM‐CSF receptor CDw116 PG, CI, SA
    G‐CSF receptor SA
    c‐kit, scf receptor CD117 CI PG
    FAS/APO‐1 CD95 PG, CI PG
    Ox40 CD134 PG
    Flt3/Flk2/STK1 CD135 PG
    ID1/ID2/MSP‐R CDw136
    4B4 CDw137 PG PG

     aAbbreviations: G‐CSF, granulocyte colony‐stimulating factor; GM‐CSF, granulocyte/macrophage colony‐stimulating factor; gp130, 130‐kDa glycoprotein signal transduction chain for IL‐6, LIF, IL‐11, etc.; IL‐1R, interleukin 1 receptor; IL‐2Rα, α subunit of interleukin 2 receptor; IFN‐γR, interferon‐γ receptor; LIF, leukemia inhibitor factor; SCF, stem cell factor; TNFR, tumor necrosis factor receptor.
     bCD numbers allocated at the Fifth and Sixth International Workshops on Human Leukocyte Differentiation Antigens (Schlossman et al., ; Ishii et al., ).
     cCommercial suppliers: BD, Becton Dickinson Immunocytometry Systems; BS, Biosource International; CI, Coulter Immunotech; EN, Endogen; GN, Genzyme; PG, Pharmingen; SA, Silenius/Amrad; ST, Serotech. New antibodies are becoming available all the time; check with the major suppliers if necessary.
  • Isotype‐matched negative control MAb at same concentration as anti‐receptor antibody
  • 2:1 (v/v) normal horse serum/normal human serum
  • Species‐specific biotinylated anti‐Ig antibody (e.g., horse mouse Ig–specific antibody; Vector Labs), concentration determined by titration (see protocol 5)
  • PE‐SA: phycoerythrin‐streptavidin (e.g., Caltag or Sigma), concentration determined by titration (see protocol 5)
  • Ice‐water bath
  • Tubes to fit flow cytometer uptake assembly (e.g., Falcon polystyrene 12 × 75–mm round‐bottom tubes) and rack
  • Tabletop centrifuge and rotor (e.g., Beckman TJ‐R with TH‐4 rotor), 4°C
  • Additional reagents and equipment for isolating cells with Ficoll‐Hypaque (unit 7.1), preparing flow cytometry fixative (unit 5.3; optional), and separating cell populations by flow cytometry (Chapter 7)
NOTE: To minimize loss of antigen and dissociation of antigen‐antibody complexes, keep cells and reagents cold throughout the procedure.

Alternate Protocol 1: Detection of Cytokine Receptors with Labeled Cytokines

  • Biotinylated cytokine (e.g., R & D Systems, Genzyme, or Pharmingen), concentration determined by titration (see protocol 4)
  • Additional reagents and equipment for labeling protein with biotin (unit 5.3; if needed)
NOTE: To minimize loss of antigen and dissociation of antigen‐antibody complexes, keep cells and reagents cold throughout the procedure.

Alternate Protocol 2: Use of Multiparameter Analysis to Detect Cytokine Receptor Expression by Lymphocyte Subpopulations

  • Normal mouse serum or 1 mg/ml normal mouse Ig (Sigma)
  • Second and (optionally) third MAbs conjugated with fluorescein or PE/Cy5, with specificities for lymphocyte subsets as required (e.g., Caltag, Pharmingen, Sigma, or Becton Dickinson Immunocytometry Systems)
  • Tricolor‐streptavidin (optional, in lieu of PE‐SA; Caltag)
NOTE: PE/Cy5 reagents may be used instead of phycoerythrin, but offer no advantage; they give slightly lower signal strength. They are best used in combination with PE, as second‐ or third‐color reagentsNOTE: To minimize loss of antigen and dissociation of antigen‐antibody complexes, keep cells and reagents cold throughout the procedure.

Support Protocol 1: Titration of Monoclonal Antibodies and Cytokines

  • Monoclonal antibodies (MAbs) against CD3 and CD25
  • Negative control antibody
NOTE: To minimize loss of antigen and dissociation of antigen‐antibody complexes, keep cells and reagents cold throughout the procedure.
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Figures

Videos

Literature Cited

Literature Cited
   Donahue, C.J., Chin, J., Hooley, J., Wolfgang‐Kimball, D., and Bauer, K.D. 1994. A comparison of 488 nm and 531 nm excitation for 3‐color immunofluorescence with dead cell discrimination. Cytometry 7(Suppl.):62.
   Haugland, R.P. 1994. Handbook of Fluorescent Probes and Research Chemicals, 5th ed. Molecular Probes, Eugene, Ore.
   Ishii, N., Takeshita, T., Kimura, Y., Tada, K., Kondo, M., Nakamura, M., and Sugamura, K. 1994. Expression of the IL‐2 receptor γ chain on various populations in human peripheral blood. Int. Immunol. 6:1273‐1277.
   Ishii, N., Asao, H., and Sugamura, K. 1997. The cytokine receptors: Section report. In Leucocyte Typing VI (T. Kishimoto, S. Goyert, H. Kikutani, D. Mason, M. Miyasaka, L. Moretta, T. Ohno, T.A. Springer, H. Sugawara, A.E.G.Kr. von dem Borne, and H. Zola, (eds.) pp. 793‐801. Garland Publishing, New York and London.
   Kondo, M., Takeshita, T., Ishii, N., Nakamura, M., Watanabe, S., Arai, K., and Sugamura, K. 1993. Sharing of the interleukin‐2 (IL‐2) receptor γ chain between receptors for IL‐2 and IL‐4. Science 262:1874‐1877.
   Macardle, P.J., Chen, Z., Shih, C‐Y., Huang, C‐M., Weedon, H., Sun, Q., Lopez, A.F., and Zola, H. 1996. Characterisation of human leucocytes bearing the IL‐3 receptor. Cell. Immunol. 168:59‐68.
   Miyawaki, T., Uehara, T., Nibu, R., Tsuji, T., Yachie, A., Yonehara, S., and Taniguchi, N. 1992. Differential expression of apoptosis‐related Fas antigen on lymphocyte subpopulations in human peripheral blood. J. Immunol. 149:3753‐3758.
   Nicola, N.A. and Metcalf, D. 1988. Binding, internalization, and degradation of 125I‐multipotential colony‐stimulating factor (interleukin‐3) by FDCP‐1 cells. Growth Fact. 1:29‐39.
   Noguchi, M., Nakamura, Y., Russell, S.M., Ziegler, S.F., Tsang, M., Cao, X., and Leonard, W.J. 1993. Interleukin‐2 receptor γ chain: A functional component of the interleukin‐7 receptor. Science 262:1877‐1880.
   Olweus, J., Lund Johansen, F., Hoffman, R., and Terstappen, L.W.M.M. 1995. Changes of surface expression of cytokine receptors during lineage commitment of early haematopoietic progenitor cells. In Leucocyte Typing V: White Cell Differentiation Antigens S. Schlossman, L. Boumsell, W. Gilks, J. Harlan, T. Kishimoto, C. Morimoto, J. Ritz, S. Shaw, R. Silverstein, T. Springer, T. Tedder, and T. Todd, eds.) pp. 1943‐1945. Oxford University Press, Oxford.
  (Schlossman, S., Boumsell, L., Gilks, W., Harlan, J., Kishimoto, T., Morimoto, C., Ritz, J., Shaw, S., Silverstein, R., Springer, T., Tedder, T., and Todd, T. eds.) 1995. Leucocyte Typing V: White Cell Differentiation Antigens. Oxford University Press, Oxford.
   Sheldon, A., Flego, L., and Zola, H. 1993. Coexpression of IL‐2 receptor p55 and p75 by circulating blood lymphocytes. J. Leukocyte Biol. 54:161‐167.
   Taga, T. and Kishimoto, T. 1992. Cytokine receptors and signal transduction. FASEB J. 6:3387‐3396.
   Weber‐Nordt, R., Meraz, M.A., and Schreiber, R.D. 1994. Lipopolysaccharide‐dependent induction of IL‐10 receptor expression on murine fibroblasts. J. Immunol. 153:3734‐3744.
   Zola, H. and Flego, L. 1992. Expression of interleukin‐6 receptor on blood lymphocytes without in vitro activation. Immunology 76:338‐340.
   Zola, H., Mantzioris, B.X., Webster, J., and Kette, F.E. 1989. Circulating human T and B lymphocytes express the p55 interleukin‐2 receptor molecule (TAC, CD25). Immunol. Cell Biol. 67:233‐237.
   Zola, H., Neoh, S.H., Mantzioris, B.X., Webster, J., and Loughnan, M.S. 1990a. Detection by immunofluorescence of surface molecules present in low copy numbers. High sensitivity staining and calibration of flow cytometer. J. Immunol. Methods 135:247‐255.
   Zola, H., Purling, R.J., Koh, L.Y., and Tsudo, M. 1990b. Expression of the p70 chain of the IL‐2 receptor on human lymphoid cells: Analysis using a monoclonal antibody and high‐sensitivity immunofluorescence. Immunol. Cell Biol. 68:217‐224.
   Zola, H., Flego, L., and Sheldon, A. 1992. Detection of cytokine receptors by high‐sensitivity immunofluorescence/flow cytometry. Immunobiology 185:350‐365.
   Zola, H., Flego, L., and Weedon, H. 1993a. Expression of IL‐4 receptor on human T and B lymphocytes. Cell. Immunol. 150:149‐158.
   Zola, H., Flego, L., and Weedon, H. 1993b. Expression of membrane receptor for tumour necrosis factor on human blood lymphocytes. Immunol. Cell Biol. 71:281‐288.
   Zola, H., Fusco, M., Flego, L., Donohoe, P.J., and Macardle, P.J. 1995. Expression of the cytokine receptor panel antibodies: High‐sensitivity immunofluorescence studies on unstimulated cells. In Leucocyte Typing V: White Cell Differentiation Antigens (F. Schlossman, L. Boumsell, W. Gilks, J. Harlan, T. Kishimoto, C. Morimoto, J. Ritz, S. Shaw, R. Silverstein, T. Springer, T. Tedder, and T. Todd, eds.) pp. 1939‐1941. Oxford University Press, Oxford.
Key Reference
   Zola et al., 1990a. See above.
   Demonstrated the sensitivity of three‐layer immunofluorescence by essentially the procedures described here. Comparison with radioligand binding studies showed that the method is capable of detecting receptor concentrations of ∼100 molecules per cell.
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