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Measurement of T and B Cell Turnover with Bromodeoxyuridine

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

¹The Scripps Research Institute, La Jolla, California
²NIAID, 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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Basic Protocol: BrdU Incorporation to Determine T and B Cell Turnover
Alternate Protocol: 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: 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: 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 (Basic Protocol), 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

Figure 4.7.1

BrdU labeling of thymocytes. Thymocytes from adult C57BL/6 mice, either untreated or given BrdU in drinking water for 7 days, were stained for CD4, CD8, and BrdU. CD4 vs. CD8 staining of total thymocytes from (A) an untreated and (B) a BrdU-treated mouse is illustrated. Histograms show BrdU staining of CD4⁺CD8⁺ thymocytes (boxed area in A and B) for (C) the untreated and (D) the BrdU-treated mouse. >99% of CD4⁺CD8⁺ cells from the BrdU-treated mouse are BrdU+.

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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.