High‐Sensitivity Immunofluorescence/Flow Cytometry: Detection of Cytokine Receptors and Other Low‐Abundance Membrane Molecules

Heddy Zola1

1 Child Health Research Institute, North Adelaide, Australia
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.3
DOI:  10.1002/0471142956.cy0603s30
Online Posting Date:  November, 2004
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Cell marker identification by traditional phenotyping techniques is now considered straight‐forward and relatively uncomplicated. In immunofluorescence/flow cytometry, the sensitivity, or detection limit, depends on the reagents, staining, and instrument parameters. The sensitivity of the most commonly used procedures, based on fluorescein‐conjugated antibodies, ∼2000 molecules of target antigen per cell, which is adequate for most of the widely used leukocyte markers. However, measuring target antigens of low density has proven very difficult indeed. Flow cytometric immunofluorescence is capable of detecting 100 molecules of target antigen per cell in practical applications, provided that every step of the staining and analysis procedure is optimized for sensitivity. This level of sensitivity reveals staining not seen using conventional analytical procedures. This unit discusses the underlying principles of high‐sensitivity immunofluorescence and provides an excellent series of protocols for the practical detection of as few as 100 target antigen molecules per cell.

Keywords: flow cytometry; sensitivity; cytokine receptors; weak signal amplification

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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Single‐Color Staining and Analysis
  • Alternate Protocol 1: Multicolor Staining and Analysis
  • Support Protocol 1: Titration and Quality Control of Monoclonal Antibodies and Detection Reagents
  • Support Protocol 2: Cell Sorting with High Sensitivity
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Single‐Color Staining and Analysis

  • Cell suspension: heparinized whole blood or Ficoll‐Hypaque‐purified cells (mononuclear cell fraction or suspension of tissue cells; units 5.1& 9.7)
  • DPBS/azide, ice cold: Dulbecco's PBS (unit 9.8) containing 0.02 M sodium azide (or other isotonic buffer with same concentration of azide)
  • Pretitrated monoclonal antibody (MAb) specific for antigen of interest (see protocol 3) in DPBS/azide, ice cold
  • Isotype control in DPBS/azide at same concentration as MAb of interest, ice cold
  • 2:1 (v/v) normal horse serum/normal human serum (or normal serum from appropriate species mixed with normal human serum; see step below)
  • Pretitrated biotinylated anti–mouse Ig (e.g., Vector Labs; see protocol 3 for titration) in DPBS/azide, ice cold
  • Pretitrated phycoerythrin‐streptavidin (PE‐SA; Caltag Labs; Sigma; see protocol 3 for titration) in DPBS/azide, ice cold
  • Lysing solution (Becton Dickinson Immunocytochemistry)
  • 12 × 75–mm (3‐ml) flow cytometry tubes
  • Refrigerated centrifuge (ideally with a 64‐tube‐capacity rotor)
  • Additional reagents and equipment for counting cells ( appendix 3A) and flow cytometry (see Chapter 1)
NOTE: Sodium azide reacts with copper in plumbing to form an explosive compound. Check institutional safety regulations concerning disposal of azide‐containing liquids.NOTE: Maintain cells cold throughout by keeping reaction tubes and reagents (except normal horse serum/normal human serum) in melting ice and setting the centrifuge at 4°C.

Alternate Protocol 1: Multicolor Staining and Analysis

  • Normal mouse serum or 1 mg/ml normal mouse Ig (Sigma) in DPBS/azide (store refrigerated), ice cold
  • Pretitrated, direct fluorochrome‐conjugated, cell‐specific MAb(s) (see introduction; see protocol 3 for titration)
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Literature Cited

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   Bikoue, A., George, F., Poncelet, P., Mutin, M., Janossy, G., and Sampol, J. 1996. Quantitative analysis of leukocyte membrane antigen expression: Normal adult values. Cytometry 26:137‐147.
   Borvak, J., Chou, C.‐S., Bell, K., van Dyke, G., Zola, H., Ramilo, O., and Vitetta, E.S. 1995. Expression of CD25 defines peripheral blood mononuclear cells with productive versus latent HIV infection. J. Immunol. 155:3196‐3204.
   Dighiero, G., Bodega, E., Mayzner, R., and Binet, J.L. 1980. Individual cell‐by‐cell quantitation of lymphocyte surface membrane Ig in normal and CLL lymphocytes and during ontogeny of mouse B lymphocytes by immunoperoxidase assay. Blood 55:93‐100.
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   Mavrangelos, C., Swart, B., Nobbs, S., Nicholson, I.C., Macardle, P.J., and Zola, H. 2004. Detection of low‐abundance membrane markers by immunofluorescence: A comparison of alternative high‐sensitivity methods and reagents. J. Immunol. Methods 289:169‐78.
   Nicholson, I.C., Lenton, K.A., Little, D.J., DeCorso, T., Lee, F.T., Scott, A.M., Zola, H., and Hohmann, A.W. 1997. Construction and characterization of a functional single chain Fv fragment for immunotherapy of B lineage leukemia and lymphoma. Mol. Immunol. 34:1157‐1165.
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   Schlossman, S.F., Boumsell, L., Gilks, W., Harlan, J.M., Kishimoto, T., Morimoto, C., Ritz, J., Shaw, S., Silverstein, R., Springer, T.A., Tedder, T.F., and Todd, R.F. 1995. Leucocyte Typing V: White cell differentiation antigens. Oxford University Press, Oxford.
   Taga, T. and Kishimoto, T. 1992. Cytokine receptors and signal transduction. FASEB J. 6:3387‐3396.
   Zola, H. 1995. Use of flow cytometry to detect cytokine receptors. In Current Protocols in Immunology (R. Coico, J.E. Coligan, E.M. Shevach, D.H. Margulies, W. Strober, and A.M. Kruisbeek, eds.) pp. 6.21.1‐6.21.24. John Wiley & Sons, New York.
   Zola, H. and Flego, L. 1992. Expression of interleukin‐6 receptor on blood lymphocytes without in vitro activation. Immunology 76:338‐340.
   Zola, H., Flego, L., and Weedon, H. 1993a. Expression of membrane receptor for tumor necrosis factor on human blood lymphocytes. Immunol. Cell Biol. 71:281‐288.
   Zola, H., Flego, L., and Weedon, H. 1993b. Expression of IL‐4 receptor on human T and B lymphocytes. Cell Immunol. 150:149‐158.
   Zola, H., Neoh, S.H., Mantzioris, B.X., Webster, J., and Loughnan, M.S. 1990. 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., Siderius, N., Flego, L., Beckman Coulter, I.G.R., and Seshadri, R. 1994. Cytokine receptor expression in leukemic cells. Leuk. Res. 18:65‐73.
   Zola, H., Fusco, M., Macardle, P.J., Flego, L., and Roberton, D. 1995. Expression of cytokine receptors by human cord blood lymphocytes—comparison with adult lymphocytes. Pediatr. Res. 38:397‐403.
Key Reference
   Zola et al., 1990. See above.
  This paper describes the high‐sensitivity immunofluorescence procedure in detail and shows typical results.
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