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.im0702s112
Online Posting Date:  February, 2016
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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. It should be noted that use of the rosetting technique to obtain purified T cells or purified non‐T cells by negative selection has largely been superceded by other techniques such as panning and immunomagnetic beads. © 2016 by John Wiley & Sons, Inc.

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: T Cell 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: T Cell Rosetting with Neuraminidase‐Treated Sheep Red Blood Cells

  Materials
  • Sheep red blood cells (SRBC; UNIT 3.8, Bondada and Robertson, ) in Alsevers solution ( appendix 22)
  • Hanks balanced salt solution (HBSS; appendix 22)
  • 1 U/ml neuraminidase (lyophilized powder, type X; Sigma‐Aldrich, cat. no. N2133) in sterile PBS ( appendix 22; store solution in 1‐ml aliquots at −20°C)
  • Complete RPMI‐10 medium ( appendix 22)
  • Peripheral blood mononuclear cells (PBMC) in complete RPMI‐10 medium
  • (1 × 107 cells/ml; unit 7.1, Fuss et al., )
  • FBS (heat‐inactivated 1 hr at 56°C)
  • Ficoll‐Hypaque solution (unit 7.1, Fuss et al., )
  • Sterile distilled water or ACK lysing buffer (UNIT 3.1, Kruisbeek, )
  • 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 (also see protocol 1Basic Protocol)
  • 2‐Aminoethylisothiouronium bromide (AET) solution
  • Phosphate‐buffered saline (PBS; appendix 22)
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Figures

Videos

Literature Cited

Literature Cited
  Bondada, S. and Robertson, D.A. 2003. Assays for B lymphocyte function. Curr. Protoc. Immunol. 56:3.8.1‐3.8.24. doi: 10.1002/0471142735.im0308s56
  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 50:39‐49. doi: 10.1016/0022‐1759(82)90302‐7
  Fuss, I.J., Kanof, M.E., Smith, P.D. and Zola, H. 2009. Isolation of whole mononuclear cells from peripheral blood and cord blood. Curr. Protoc. Immunol. 85:7.1.1‐7.1.8. doi: 10.1002/0471142735.im0701s85
  Heine, G., Sims, G.P., Worm, M., Lipsky, P.E. and Radbruch, A. 2011. Isolation of human B cell populations. Curr. Protoc. Immunol. 94:7.5.1‐7.5.14. doi: 10.1002/0471142735.im0705s94
  Horgan, K., Shaw, S. and Boirivant, M. 2009. Immunomagnetic purification of T cell subpopulations. Curr. Protoc. Immunol. 85:7.4.1‐7.4.9. doi: 10.1002/0471142735.im0704s85
  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. doi: 10.1016/0022‐1759(80)90164‐7
  Kanof, M.E. 1991. Purification of T cell subpopulations. Curr. Protoc. Immunol. 00:7.3.1‐7.3.5. doi: 10.1002/0471142735.im0703s00
  Kruisbeek, A.M. 2001. Isolation of mouse mononuclear cells. Curr. Protoc. Immunol. 39:3.1.1‐3.1.5. doi: 10.1002/0471142735.im0301s39
  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. doi: 10.1016/0022‐1759(79)90239‐4
  Mehrishi, J.N. and Bakacs, T. 2013. A novel method of CD34+ cell separation from umbilical cord blood. Transfusion 53:2675‐2680. doi: 10.1111/trf.12123
  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.
  Strober, W. 2012. Immunologic studies in humans. Curr. Protoc. Immunol. 97:7.0.1‐7.0.8. doi: 10.1002/0471142735.im0700s97
  Wahl, L.M., Wahl, S.M., Smythies, L.E. and Smith, P.D. 2006. Isolation of human monocyte populations. Curr. Protoc. Immunol. 70:7.6A.1‐7.6A.10. doi: 10.1002/0471142735.im0706as70
  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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