Abstract
Lymphocyte characterization is primarily based on the differential expression of surface markers. In this context, flow-cytometry analysis (FACS) is an exceptional technique that not only allows the identification of B-cell subsets, but can also be used to evaluate cell function, activation, and division. Here, we will combine the use of FACS analysis and ELISA techniques to identify murine bone marrow and peripheral B-cell subsets. The main function of B cells, derived through a multistage differentiation process from precursor cells, is to produce antibodies. This task is performed by terminally differentiated B cells called antibody-secreting cells (ASC) present at mucosal sites, in the bone marrow and in the spleen. The number and specificity of ASC can be measured by Enzyme-linked immunosorbent spot (ELISPOT) assay, a variation of the enzyme-linked immunosorbent assay (ELISA) used to quantify serum immunoglobulins.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Godin I, Cumano A (2002) The hare and the tortoise: an embryonic haematopoietic race. Nat Rev 2(8):593–604
Ghosn EE, Sadate-Ngatchou P, Yang Y, Herzenberg LA, Herzenberg LA (2011) Distinct progenitors for B-1 and B-2 cells are present in adult mouse spleen. Proc Natl Acad Sci USA 108(7):2879–2884
Rosado MM, Aranburu A, Capolunghi F, Giorda E, Cascioli S, Cenci F, Petrini S, Miller E, Leanderson T, Bottazzo GF, Natali PG, Carsetti R (2009) From the fetal liver to spleen and gut: the highway to natural antibody. Mucosal Immunol 2(4):351–361
Carsetti R, Kohler G, Lamers MC (1995) Transitional B cells are the target of negative selection in the B cell compartment. J Exp Med 181(6):2129–2140
Carsetti R, Rosado MM, Wardmann H (2004) Peripheral development of B cells in mouse and man. Immunol Rev 197:179–191
Quah BJ, Warren HS, Parish CR (2007) Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester. Nat Protoc 2(9):2049–2056
Hardy RR, Carmack CE, Shinton SA, Kemp JD, Hayakawa K (1991) Resolution and characterisation of pro-B and Pre-Pro-B cell stages in normal mouse bone marrow. J Exp Med 173:1213–1225
Hardy RR, Li YS, Allman D, Asano M, Gui M, Hayakawa K (2000) B-cell commitment, development and selection. Immunol Rev 173:23–32
Rolink A, Grawunder U, Winkler TH, Karasuyama H, Melchers F (1994) IL-2 receptor alpha chain (CD25, TAC) expression defines a crucial stage in pre-B cell development. Int Immunol 6(8):1257–1264
Kitamura D, Roes J, Kuhn R, Rajewsky KA (1991) B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin mu chain gene. Nature 350(6317):423–426
Casola S, Otipoby KL, Alimzhanov M, Humme S, Uyttersprot N, Kutok JL, Carroll MC, Rajewsky K (2004) B cell receptor signal strength determines B cell fate. Nat Immunol 5(3):317–327
Chen X, Martin F, Forbush KA, Perlmutter RM, Kearney JF (1997) Evidence for selection of a population of multi-reactive B cells into the splenic marginal zone. Int Immunol 9(1):27–41
Hayakawa K, Hardy RR, Honda M, Herzenberg LA, Steinberg AD, Herzenberg LA (1984) Ly-1 B cells: functionally distinct lymphocytes that secrete IgM autoantibodies. Proc Natl Acad Sci USA 81(8):2494–2498
Baumgarth N (2011) The double life of a B-1 cell: self-reactivity selects for protective effector functions. Nat Rev Immunol 11(1):34–46
Hardy RR, Kincade PW, Dorshkind K (2007) The protean nature of cells in the B lymphocyte lineage. Immunity 26(6):703–714
Rolink AG, Andersson J, Melchers F (2004) Molecular mechanisms guiding late stages of B-cell development. Immunol Rev 197:41–50
Acknowledgments
We thank Dr. Claudio Pioli for his critical review of the manuscript. R.C. is supported by EUROPAD NET (EC Grant Nr. 201549).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Rosado, M.M., Scarsella, M., Cascioli, S., Giorda, E., Carsetti, R. (2013). Evaluating B-Cells: From Bone Marrow Precursors to Antibody-Producing Cells. In: Allen, I. (eds) Mouse Models of Allergic Disease. Methods in Molecular Biology, vol 1032. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-496-8_4
Download citation
DOI: https://doi.org/10.1007/978-1-62703-496-8_4
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-495-1
Online ISBN: 978-1-62703-496-8
eBook Packages: Springer Protocols