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

  • Basic Protocol 1: Isolation of T Cell Populations by Indirect Panning
  • Alternate Protocol 1: Isolation of T Cell Populations by Antibody/Complement–Mediated Cytotoxicity
  • Commentary
     
 
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Materials

Basic Protocol 1: 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 1: 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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