Analysis of B-Cell Life-Span and Homeostasis

  • Irmgard Föster
Part of the Methods in Molecular Biology book series (MIMB, volume 271)


In the past, the life-span of B cells in rodents has been determined by a variety of methods, leading to conflicting results. Among the various techniques employed, labeling of dividing cells with the thymidine analog 5-bromo-2′-deoxyuridine (BrdU) has turned out to be a versatile and reliable procedure. Labeling of the cells can be easily performed in vivo by feeding BrdU in the drinking water for extended periods of time or by an ip injection of BrdU for short-term labeling experiments. Using the protocol described, it is possible to combine flow cytometric detection of incorporated BrdU simultaneously with fluorescence staining of various cell surface markers.

Key Words

BrdU B cell life-span flow cytometry proliferation 


  1. 1.
    Osmond, D. G. (1993) The turnover of B-cell populations. Immunol. Today 14, 34–37.PubMedCrossRefGoogle Scholar
  2. 2.
    Freitas, A. A. and Rocha, B. B. (1993) Lymphocyte lifespans: homeostasis, selection and competition. Immunol. Today 14, 25–29.PubMedCrossRefGoogle Scholar
  3. 3.
    Rajewsky, K. (1993) B-cell lifespans in the mouse—why to debate what? Immunol. Today 14, 40, 41; discussion 41–43.PubMedCrossRefGoogle Scholar
  4. 4.
    Sprent, J. and Basten, A. (1973) Circulating T and B lymphocytes of the mouse, II: lifespan. Cell Immunol. 7, 40–59.PubMedCrossRefGoogle Scholar
  5. 5.
    Röke, C., Hougen, H. P., and Everett, N. B. (1975) Long-lived T and B lymphocytes in the bone marrow and thoracic duct lymph of the mouse. Cell Immunol. 15, 82–93.CrossRefGoogle Scholar
  6. 6.
    Röke, C. and Everett, N. B. (1975) Life span of small lymphocytes in the thymolymphatic tissues of normal and thymus-deprived BALB/C mice. Anat. Rec. 183, 83–94.CrossRefGoogle Scholar
  7. 7.
    Opstelten, D. and Osmond, D. G. (1983) Pre-B cells in mouse bone marrow: immunofluorescence stathmokinetic studies of the proliferation of cytoplasmic µ-chain-bearing cells in normal mice. J. Immunol. 131, 2635–2640.PubMedGoogle Scholar
  8. 8.
    Freitas, A. A., Rocha, B., Forni, L., and Coutinho, A. (1982) Population dynamics of B lymphocytes and their precursors: demonstration of high turnover in the central and peripheral lymphoid organs. J. Immunol. 128, 54–60.PubMedGoogle Scholar
  9. 9.
    Heyman, R. A., Borrelli, E., Lesley, J., et al. (1989) Thymidine kinase obliteration: creation of transgenic mice with controlled immune deficiency. Proc. Natl. Acad. Sci. USA 86, 2698–2702.PubMedCrossRefGoogle Scholar
  10. 10.
    Gratzner, H. G. (1982) Monoclonal antibody to 5-bromo-and 5-iododeoxyuridine: a new reagent for detection of DNA replication. Science 218, 474, 475.PubMedCrossRefGoogle Scholar
  11. 11.
    Houck, D. W. and Loken, M. R. (1985) Simultaneous analysis of cell surface antigens, bromodeoxyuridine incorporation and DNA content. Cytometry 6, 531–538.PubMedCrossRefGoogle Scholar
  12. 12.
    Föster, I., Vieira, P., and Rajewsky, K. (1989) Flow cytometric analysis of cell proliferation dynamics in the B cell compartment of the mouse. Int. Immunol. 1, 321–331.CrossRefGoogle Scholar
  13. 13.
    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.PubMedGoogle Scholar
  14. 14.
    Penit, C. and Vasseur, F. (1993) Phenotype analysis of cycling and postcycling thymocytes: evaluation of detection methods for BrdUrd and surface proteins. Cytometry 14, 757–763.PubMedCrossRefGoogle Scholar
  15. 15.
    Tough, D. F. and Sprent, J. (1994) Turnover of naive-and memory-phenotype T cells. J. Exp. Med. 179, 1127–1135.PubMedCrossRefGoogle Scholar
  16. 16.
    Kaufman, E. R. and Davidson, R. L. (1978) Bromodeoxyuridine mutagenesis in mammalian cells: mutagenesis is independent of the amount of bromouracil in DNA. Proc. Natl. Acad. Sci. USA 75, 4982–4986.PubMedCrossRefGoogle Scholar
  17. 17.
    Tapscott, S. J., Lassar, A. B., Davis, R. L., and Weintraub, H. (1989) 5-bromo-2′-deoxyuridine blocks myogenesis by extinguishing expression of MyoD1. Science 245, 532–536.PubMedCrossRefGoogle Scholar
  18. 18.
    Gray, D. (1988) Population kinetics of rat peripheral B cells. J. Exp. Med. 167, 805–816.PubMedCrossRefGoogle Scholar
  19. 19.
    Föster, 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. USA 87, 4781–4784.CrossRefGoogle Scholar
  20. 20.
    Schittek, B., Rajewsky, K., and Föster, I. (1991) Dividing cells in bone marrow and spleen incorporate bromodeoxyuridine with high efficiency. Eur. J. Immunol. 21, 235–238.PubMedCrossRefGoogle Scholar
  21. 21.
    Schittek, B. and Rajewsky, K. (1990) Maintenance of B-cell memory by long-lived cells generated from proliferating precursors. Nature 346, 749–751.PubMedCrossRefGoogle Scholar
  22. 22.
    Tough, D. F. and Sprent, J. (1995) Lifespan of lymphocytes. Immunol. Res. 14, 1–12.PubMedCrossRefGoogle Scholar
  23. 23.
    Penit, C. and Vasseur, F. (1997) Expansion of mature thymocyte subsets before emigration to the periphery. J. Immunol. 159, 4848–4856.PubMedGoogle Scholar
  24. 24.
    Kamath, A. T., Henri, S., Battye, F., Tough, D. F., and Shortman, K. (2002) Developmental kinetics and lifespan of dendritic cells in mouse lymphoid organs. Blood 100, 1734–1741.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  • Irmgard Föster
    • 1
  1. 1.Institute for Medical Microbiology, Immunology, and Hygiene and Department of Internal Medicine IITechnical University of MunichMunichGermany

Personalised recommendations