In Vivo Depletion of T Lymphocytes

Karen Laky1, Ada M. Kruisbeek2

1 National Institutes of Health, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, 2 Netherlands Cancer Institute, Amsterdam
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 4.1
DOI:  10.1002/0471142735.im0401s113
Online Posting Date:  April, 2016
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In vivo depletion of T lymphocytes is a means of studying the role of specific T cell populations during defined phases of in vivo immune responses. In this unit, a protocol is provided for injecting monoclonal antibodies (mAbs) into wild‐type adult mice. Depletion of the appropriate subset of cells is verified by flow cytometry analysis of lymph node and spleen cell suspensions in pilot experiments. Once conditions have been established, depleted mice can be used to study the impact of T cell subsets on a variety of in vivo immune responses. The depleted condition may be maintained by repeated injections of the monoclonal antibody, or reversed by normal thymopoiesis following discontinuation of antibody administration. © 2016 by John Wiley & Sons, Inc.

Keywords: in vivo; monoclonal antibody; depletion; T cells

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Table of Contents

  • Basic Protocol 1:  
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:  

  • Adult mice (>6 weeks old)
  • mAb for injection (see Reagents and Solutions): may be commercially purchased or purified from ascites fluid or tissue culture supernatant (unit 2.7; Andrew and Titus, ); mAb preparations should be sterile, azide‐free, and at a concentration of 1 to 2 mg/ml in 1 × HBSS ( appendix 2A), PBS ( appendix 2A), or normal saline (0.9% NaCl)
  • HBSS ( appendix 2A), PBS ( appendix 2A), or normal saline (0.9% w/v NaCl), sterile ( appendix 2A)
  • Fluorescently labeled mAb for flow cytometric analyses to verify depletion (see Reagents and Solutions)
  • 1‐ml sterile syringes with 25‐ to 27‐G needles
  • 4‐ml round‐bottom polystyrene tubes (e.g., Corning Falcon)
  • Additional reagents and equipment for intraperitoneal injection of mice (unit 1.6; Donovan and Brown, ), euthanasia of mice (unit 1.8), removal of lymphoid organs (unit 1.9; Reeves and Reeves, ), preparing single‐cell suspensions from spleen and lymph node (unit 3.1; Kruisbeek, ), staining cells (unit 5.3; Holmes et al., ), and flow cytometry analysis (unit 5.4; Holmes et al., )
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Literature Cited

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