Purification of T Cell Subpopulations

Marjorie E. Kanof1

1 Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.3
DOI:  10.1002/0471142735.im0703s00
Online Posting Date:  May, 2001
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Abstract

This unit describes a procedure for isolating T cell populations or subpopulations using the method of indirect panning. In this method, cells are selected by their capacity to bind to antibody-coated plates on the basis of particular cell-surface markers. It is superior to the antibody/complement lysis method (also presented) because the nonselected cell population can be retrieved.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol: Isolation of T Cell Populations by Indirect Panning
  • Alternate Protocol: Isolation of T Cell Populations by Antibody/Complement–Mediated Cytotoxicity
  • Commentary
  • Bibliography
     
 
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Materials

Basic Protocol: Isolation of T Cell Populations by Indirect Panning

 Materials
  • Affinity-purified and human-Ig-absorbed goat anti-mouse Ig (Tago #4150)
  • 0.05 M Tris×Cl, pH 9.5
  • Specific mouse antibodies (polyclonal or monoclonal antibody; e.g., Coulter, or Becton Dickinson)
  • Phosphate-buffered saline (PBS; appendix 2A)
  • T cell population (unit 7.2)
  • 5% and 1% heat-inactivated (1 hr, 56°C) FCS in PBS
  • Complete RPMI medium (serum-free and filter sterilized; appendix 2A)
  • 15 × 100–mm plastic petri dish, bacteriological grade (not treated for tissue culture)
  • 15-ml centrifuge tube
  • Beckman GPR centrifuge with GH-3.7 horizontal rotor (or equivalent temperature-controlled centrifuge)
  • Flat surface at 4°C
  • Inverted microscope
  • Additional reagents and equipment for immunofluorescent staining (unit 5.3) and flow cytometry (units 5.4 & 7.9)

Alternate Protocol: Isolation of T Cell Populations by Antibody/Complement–Mediated Cytotoxicity

 Additional Materials
  • Baby rabbit complement (Cedarlane Laboratories)
  • Complete RPMI-1 medium (appendix 2A)
  • Additional reagents and equipment for cell counting and viability staining (appendix 3A) and Ficoll-Hypaque cell separation (unit 7.1)
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Figures

Videos

Literature Cited

 Literature Cited
    Bianco, C., Patrick, C.M., and Nussenzweig, V. 1970. A population of lymphocytes bearing a membrane receptor for antigen-antibody complement complexes. I. Separation and characterization. J. Exp. Med. 132:702-720.
    Engleman, E.G., Benike, C.J., Grumet, F.C., and Evans, R.L. 1982. Activation of human T lymphocyte subsets: Helper and suppressor/ cytotoxic T cells recognize and respond to distinct histocompatibility antigens. J.Immunol. 127:2124-2129.
    Mage, M.G., McHugh, L.L., and Rothstein, T.L. 1977. Mouse lymphocytes with and without surface immunoglobulins: Preparative scale separation on polystyrene tissue culture dishes coated with specifically purified anti-immunoglobulin. J. Immunol. Methods 15:47-56.
    Padmanabhan, R., Corsico, C.D., Howard, T.H., Holter, W., Fordis, C.M., Willingham, M., and Howard, B.H. 1980. Purification of transiently transfected cells by magnetic affinity cell sorting. Anal. Biochem. 170:341-348.
    Payne, S.M., Sharrow, S.O., Shearer, G.M., and Biddison, W.E. 1981. Preparative separation of human T cells reactive with the OKT4 monoclonal antibody. Int. J. Immunopharmocol. 3:227-232.
 Key Reference
    Wysocki, L.J. and Sato, V.L. 1978. “Panning” for lymphocytes: A method for cell separation. Proc. Nat. Acad. Sci. U.S.A. 75:284-2848.

Reports in detail the procedure for both direct and indirect panning.

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