Isolation of T Cells Using Rosetting Procedures

Marjorie E. Kanof1

1 Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.2
DOI:  10.1002/0471142735.im0702s19
Online Posting Date:  May, 2001
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Abstract

This unit describes a procedure for separating T cells from other mononuclear cells by exploiting the unique ability of cells to bind to and form rosettes with sheep red blood cells (SRBC). This isolation method also allows recovery of the nonrosetting cell population (B lymphocytes, monocytes, and macrophages). Neuraminidase‐ and 2 ‐aminoethylisothiouronium bromide (AET)‐treated SRBC are used for rosetting because of enhanced binding to T cells.

     
 
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Table of Contents

  • Basic Protocol 1: Rosetting with Neuraminidase‐Treated Sheep Red Blood Cells
  • Alternate Protocol 1: Rosetting with AET‐Treated Sheep Red Blood Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Rosetting with Neuraminidase‐Treated Sheep Red Blood Cells

  Materials
  • Sheep red blood cells (SRBC; unit 3.8) in recipeAlsevers solution ( appendix 2A)
  • Hanks balanced salt solution (HBSS; appendix 2A)
  • 1 U/ml neuraminidase (lyophilized powder, type X; Sigma #N2133) in sterile PBS ( appendix 2A; store solution in 1‐ml aliquots at −20°C)
  • Complete RPMI‐10 medium ( appendix 2A)
  • Peripheral blood mononuclear cells (PBMC) in complete RPMI‐10 medium (1 × 107 cells/ml; unit 7.1)
  • Fetal calf serum (FCS; heat‐inactivated 1 hr at 56°C)
  • Ficoll‐Hypaque solution (unit 7.1)
  • Sterile distilled water or ACK lysing buffer (unit 3.1)
  • Beckman GPR centrifuge with GH‐3.7 horizontal rotor (or equivalent temperature‐controlled centrifuge)
  • 15 and 50‐ml conical centrifuge tubes (e.g., Falcon)
  • 15‐ml round‐bottom centrifuge tubes (e.g., Falcon)

Alternate Protocol 1: Rosetting with AET‐Treated Sheep Red Blood Cells

  Additional Materials
  • recipe2‐aminoethylisothiouronium bromide (AET) solution
  • Phosphate‐buffered saline (PBS; appendix 2A)
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Figures

  •   FigureFigure 7.2.1 Separation of SRBC from other peripheral blood components on a Ficoll‐Hypaque gradient.

Videos

Literature Cited

Literature Cited
   Falkoff, R.M., Peters, M., and Fauci, A.S. 1982. T cell enrichment and depletion of human peripheral blood mononuclear cell preparations. Unexpected findings in the study of the functional activities of the separated populations. J. Immunol. Methods 50:3999‐49.
   Indiveri, F., Huddlestone, J., Pellegrino, M.A., and Ferrone, S. 1980. Isolation of human T lymphocytes: Comparison between wool filtration and rosetting with neuraminidase (VCN) and 2‐aminoethylisothiouronium bromide (AET)‐treated sheep red blood cells. J. Immunol. Methods 34:107‐115.
   Madsen, M. and Johnson, H.E. 1979. A methodological study of E‐rosette formation using AET‐treated sheep erythrocytes. J. Immunol. Methods 27:61‐74.
   Silva, A., Lopez‐Botet, M., Alvarez, J., and De Landazuri, M.O. 1981. Enhancement of the functional activities of human T cells after their interaction with SRBC. J. Immunol. 126:393‐397.
   Weiner, M.S., Bianco, C., and Nussenzweig, V. 1973. Enhanced binding of neuraminidase treated sheep erythrocytes to human T lymphocytes. Blood 42:939‐946.
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