Abstract
This chapter provides information on the application of flow cytometry for analysis of B-cell development, describing in detail the particular surface proteins that can serve as markers for recognizing distinct stages in this process. These cell fractions range from just prior to initial heavy chain rearrangement, the germline pro-B stage, through D-J rearranged pro-B and heavy chain expressing pre-B stages, to the maturing surface BCR positive B-cell stages. It also outlines assays for the characterization of these cells, including procedures for testing functional lineage restriction, determination of rearrangement status, analyses of gene expression at the ribonucleic acid (RNA) and protein level, and assessment of cell cycle state.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Bosma, G. C., Custer, R. P., and Bosma, M. J. (1983) A severe combined immunodeficiency mutation in the mouse. Nature 301, 527–530.
Bosma, M. J. and Carroll, A. M. (1991) The SCID mouse mutant: definition, characterization and potential uses. Annu. Rev. Immunol. 9, 323–350.
Blunt, T., Gell, D., Fox, M., et al. (1996) Identification of a nonsense mutation in the carboxyl-terminal region of DNA-dependent protein kinase catalytic subunit in the SCID mouse. Proc. Natl. Acad. Sci. USA 93, 10,285–10,290.
Hardy, R. R., Kemp, J. D., and Hayakawa, K. (1989) Analysis of lymphoid population in SCID mice: detection of a potential B lymphocyte progenitor population present at normal levels in SCID mice by three color flow cytometry with B220 and S7. Curr. Top. Microbiol. Immunol. 152, 19–25.
Hardy, R. R., Carmack, C. E., Shinton, S. A., Kemp, J. D., and Hayakawa, K. (1991) Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow. J. Exp. Med. 173, 1213–1225.
Fried, M., Hardy, R. R., and Bosma, M. J. (1989) Transgenic SCID mice with a functionally rearranged immunoglobulin heavy chain gene. Curr. Top. Microbiol. Immunol. 152, 107–114.
Reichman-Fried, M., Bosma, M. J., and Hardy, R. R. (1993) B lineage cell in µ-transgenic SCID mice proliferate in response to IL-7 but fail to show evidence of immunoglobulin light chain rearrangement. Int. Immunol. 5, 303–310.
Spanopoulou, E., Roman, C. A., Corcoran, L. M., et al. (1994) Functional immunoglobulin transgenes guide ordered B-cell differentiation in Rag-1-deficient mice. Genes Dev. 8, 1030–1042.
Fang, W., Mueller, D. L., Pennell, C. A., et al. (1996) Frequent aberrant immunoglobulin gene rearrangements in pro-B cells revealed by a bcl-xL transgene. Immunity 4, 291–299.
Bain, G., Robanus Maandag, E. C., te Riele, H. P., et al. (1997) Both E12 and E47 allow commitment to the B cell lineage. Immunity 6, 145–154.
Li, Y. S., Wasserman, R., Hayakawa, K., and Hardy, R. R. (1996) Identification of the earliest B lineage stage in mouse bone marrow. Immunity 5, 527–535.
Allman, D., Li, J., and Hardy, R. R. (1999) Commitment to the B lymphoid lineage occurs before DH-JH recombination. J. Exp. Med. 189, 735–740.
Tudor, K. S., Payne, K. J., Yamashita, Y., and Kincade, P. W. (2000) Functional assessment of precursors from murine bone marrow suggests a sequence of early B lineage differentiation events. Immunity 12, 335–345.
Li, Y.-S., Hayakawa, K., and Hardy, R. R. (1993) The regulated expression of B lineage associated genes during B cell differentiation in bone marrow and fetal liver. J. Exp. Med. 178, 951–960.
Allman, D., Lindsley, R. C., DeMuth, W., Rudd, K., Shinton, S. A., and Hardy, R. R. (2001) Resolution of three nonproliferative immature splenic B cell subsets reveals multiple selection points during peripheral B cell maturation. J. Immunol. 167, 6834–6840.
Kondo, M., Weissman, I. L., and Akashi, K. (1997) Identification of clonogenic common lymphoid progenitors in mouse bone marrow. Cell 91, 661–672.
Wells, S. M., Kantor, A. B., and Stall, A. M. (1994) CD43 (S7) expression identifies peripheral B cell subsets. J. Immunol. 153, 5503–5515.
Hardy, R. R. and Hayakawa, K. (1991) A developmental switch in B lymphopoiesis. Proc. Natl. Acad. Sci. USA 88, 11,550–11,554.
Hardy, R. R., Shinton, S. A., and Hayakawa, K. (1992) Repopulation of SCID mice with fetal-derived B-lineage cells. Curr. Top. Microbiol. Immunol. 182, 73–80.
Cumano, A., Dorshkind, K., Gillis, S., and Paige, C. J. (1990) The influence of S17 stromal cells and interleukin 7 on B cell development. Eur. J. Immunol. 20, 2183–2189.
Cumano, A., Paige, C. J., Iscove, N. N., and Brady, G. (1992) Bipotential precursors of B cells and macrophages in murine fetal liver. Nature 356, 612–615.
Carleton, M., Haks, M. C., Smeele, S. A., et al. (2002) Early growth response transcription factors are required for development of CD4(−)CD8(−) thymocytes to the CD4(+)CD8(+) stage. J. Immunol. 168, 1649–1658.
Schmitt, T. M. and Zuniga-Pflucker, J. C. (2002) Induction of T-cell development from hematopoietic progenitor cells by delta-like-1 in vitro. Immunity 17, 749–756.
Kawamoto, H., Ohmura, K., and Katsura, Y. (1997) Direct evidence for the commitment of hematopoietic stem cells to T, B and myeloid lineages in murine fetal liver. Int. Immunol. 9, 1011–1019.
Dou, Y. M., Watanabe, Y., Aiba, Y., Wada, K., and Katsura, Y. (1994) A novel culture system for induction of T-cell development: modification of fetal thymus organ culture. Thymus 23, 195–207.
Ikawa, T., Kawamoto, H., Fujimoto, S., and Katsura, Y. (1999) Commitment of common T/Natural killer (NK) progenitors to unipotent T and NK progenitors in the murine fetal thymus revealed by a single progenitor assay. J. Exp. Med. 190, 1617–1626.
Schlissel, M. S. and Baltimore, D. (1989) Activation of immunoglobulin kappa gene rearrangement correlates with induction of germline kappa gene transcription. Cell 58, 1001–1007.
Gu, H., Kitamura, D., and Rajewsky, K. (1991) B cell development regulated by gene rearrangement: arrest of maturation by membrane-bound D mu protein and selection of DH element reading frames. Cell 65, 47–54.
Gu, H., Tarlinton, D., Muller, W., Rajewsky, K., and Forster, I. (1991) Most peripheral B cells in mice are ligand selected. J. Exp. Med. 173, 1357–1371.
Kitamura, D. and Rajewsky, K. (1992) Targeted disruption of mu chain membrane exon causes loss of heavy-chain allelic exclusion. Nature 356, 154–156.
Wasserman, R., Li, Y.-S., and Hardy, R. R. (1995) Differential expression of the blk and ret tyrosine kinases during B lineage development is dependent on Ig rearrangement. J. Immunol. 155, 644–651.
Wasserman, R., Li, Y. S., and Hardy, R. R. (1997) Down-regulation of terminal deoxynucleotidyl transferase by Ig heavy chain in B lineage cells. J. Immunol. 158, 1133–1338.
Wasserman, R., Li, Y. S., Shinton, S. A., et al. (1998) A novel mechanism for B cell repertoire maturation based on response by B cell precursors to pre-B receptor assembly. J. Exp. Med. 187, 259–264.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc.
About this protocol
Cite this protocol
Hardy, R.R., Shinton, S.A. (2004). Characterization of B Lymphopoiesis in Mouse Bone Marrow and Spleen. In: Gu, H., Rajewsky, K. (eds) B Cell Protocols. Methods in Molecular Biology, vol 271. Humana Press. https://doi.org/10.1385/1-59259-796-3:001
Download citation
DOI: https://doi.org/10.1385/1-59259-796-3:001
Publisher Name: Humana Press
Print ISBN: 978-1-58829-347-3
Online ISBN: 978-1-59259-796-3
eBook Packages: Springer Protocols