Measurement of T and B Cell Turnover with Bromodeoxyuridine

David F. Tough1, Jonathan Sprent1, Geoffrey L. Stephens2

1 The Scripps Research Institute, La Jolla, California, 2 NIAID, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 4.7
DOI:  10.1002/0471142735.im0407s77
Online Posting Date:  May, 2007
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Abstract

This unit describes two methods used to determine the rate of turnover of T and B cells in vivo. In each method, this is accomplished by administering 5‐bromo‐2′‐deoxyuridine (BrdU), a thymidine analog, and measuring the rate at which cells become labeled with this DNA precursor during the S phase of the cell cycle. Cells that contain BrdU are then detected in vitro using fluorochrome‐labelled anti‐BrdU monoclonal antibody by flow cytometry. The unit includes an Alternate Protocol that may be useful in situations where the Basic Protocol proves incompatible with particular antibody‐fluorochrome conjugates or other downstream staining procedures. Using this procedure, a wide range of antibody‐fluorochrome combinations can be used.

     
 
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Table of Contents

  • Basic Protocol 1: BrdU Incorporation to Determine T and B Cell Turnover
  • Alternate Protocol 1: Simultaneous Measurement of T Cell Turnover Using Hi‐Dimensional Flow Cytometry
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: BrdU Incorporation to Determine T and B Cell Turnover

  Materials
  • Experimental animals
  • 0.8 mg/ml BrdU (Sigma) in water (for oral administration) or 4 mg/ml BrdU in PBS (for injection)
  • PBS, pH 7.4 ( appendix 2A)
  • 0.15 M NaCl, ice cold
  • 95% ethanol, ice cold
  • Paraformaldehyde fixative (see recipe)
  • DNase I solution (see recipe)
  • Anti‐BrdU‐FITC (Becton Dickinson Immunocytometry)
  • 15 × 75–mm round‐bottom polystyrene tubes
  • Flow cytometer capable of 3‐ or 4‐color analysis
  • Additional reagents and equipment for removal of mouse lymphoid organs (unit 1.9), preparation of mononuclear cell suspensions (unit 3.1), cell surface staining of lymphocytes for flow cytometry (unit 5.3), and flow cytometry (unit 5.4)

Alternate Protocol 1: Simultaneous Measurement of T Cell Turnover Using Hi‐Dimensional Flow Cytometry

  Materials
  • Experimental animals
  • Staining buffer (see recipe)
  • Fixing buffer (see recipe)
  • Permeabilization buffer (see recipe)
  • DNase solution (see recipe), prewarmed to 37°C
  • Fluorochrome‐conjugated anti‐BrdU antibody (BD Biosciences)
  • 12 × 75–mm round‐bottom polystyrene tubes
  • Water bath, prewarmed to 37°C
  • Flow cytometer capable of multi‐color analysis
  • Additional reagents and equipment required for BrdU treatment ( protocol 1), the removal of mouse lymphoid tissues (unit 1.9), preparation of single cell suspensions (unit 3.1), cell‐surface staining for flow cytometry (unit 5.3), and flow cytometry (unit 5.4)
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Figures

Videos

Literature Cited

Literature Cited
   Carayon, P. and Bord, A. 1992. Identification of DNA‐replicating lymphocyte subsets using a new method to label the bromo‐deoxyuridine incorporated into the DNA. J. Immunol. Methods 147:225‐230.
   Dolbeare, F. and Gray, J.W. 1988. Use of restriction endonucleases and exonuclease III to expose halogenated pyrimidines for immunochemical staining. Cytometry 9:631‐635.
   Feinendegen, L.E. 1967. Tritium Labeled Molecules in Biology and Medicine. Academic Press, New York.
   Feinendegen, L.E., Heiniger, H.J., Friedrich, G., and Cronkite, E.P. 1973. Differences in reutilization of thymidine in hemopoietic and lymphopoietic tissues of the normal mouse. Cell Tissue Kinet. 6:573‐585.
   Forster, I. and Rajewsky, K. 1990. The bulk of the peripheral B‐cell pool in mice is stable and not rapidly renewed from the bone marrow. Proc. Natl. Acad. Sci. U.S.A. 87:4781‐4784.
   Gonchoroff, N.J., Katzmann, J.A., Currie, R.M., Evans, E.L., Houck, D.W., Kline, B.C., Greipp, P.R., and Loken, M.R. 1986. S‐phase detection with an antibody to bromodeoxyuridine. Role of DNase pretreatment. J. Immunol. Methods 93:97‐101.
   Gratzner, H.G. 1982. Monoclonal antibody to 5‐bromo‐ and 5‐iododeoxyuridine: A new reagent for detection of DNA replication. Science 218:474‐475.
   Houck, D.W. and Loken, M.R. 1985. Simultaneous analysis of cell surface antigens, bromodeoxyuridine incorporation and DNA content. Cytometry 6:531‐538.
   Kriss, J.P. and Revesz, L. 1962. The distribution and fate of bromodeoxyuridine and bromodeoxycytidine in the mouse and rat. Cancer Res. 22:254‐265.
   Lucas, B., Vasseur, F., and Penit, C. 1994. Production, selection, and maturation of thymocytes with high surface density of TCR. J. Immunol. 153:53‐62.
   Penit, C. and Papiernik, M. 1986. Regulation of thymocyte proliferation and survival by deoxynucleosides. Deoxycytidine produced by thymic accessory cells protects thymocytes from deoxyguanosine toxicity and stimulates their spontaneous proliferation. Eur. J. Immunol. 16:257‐263.
   Schittek, B., Rajewsky, K., and Forster, I. 1991. Dividing cells in bone marrow and spleen incorporate bromodeoxyuridine with high efficiency. Eur. J. Immunol. 21:235‐238.
   Tough, D.F. and Sprent, J. 1994. Turnover of naive‐ and memory‐phenotype T cells. J. Exp. Med. 179:1127‐1135.
   Van Furth, R. and Van Zwet, T.L. 1988. Immunocytochemical detection of 5‐bromo‐2‐deoxyuridine incorporation in individual cells. J. Immunol. Methods 108:45‐51.
   Westermann, J., Ronneberg, S., Fritz, F.J., and Pabst, R. 1989. Proliferation of lymphocyte subsets in the adult rat: A comparison of different lymphoid organs. Eur. J. Immunol. 19:1087‐1093.
   Wynford‐Thomas, D. and Williams, E.D. 1986. Use of bromodeoxyuridine for cell kinetic studies in intact animals. Cell Tissue Kinet. 19:179‐182.
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