Isolation and Analysis of B-cell Progenitors from Bone Marrow by Flow Cytometry - PubMed (original) (raw)

Isolation and Analysis of B-cell Progenitors from Bone Marrow by Flow Cytometry

Hongchang Zhao et al. Bio Protoc. 2023.

Abstract

B cells play a critical role in host defense, producing antibodies in response to microbial infection. An inability to produce an effective antibody response leaves affected individuals prone to serious infection; therefore, proper B-cell development is essential to human health. B-cell development begins in the bone marrow and progresses through various stages until maturation occurs in the spleen. This process involves several sequential, complex events, starting with pre- and pro-B cells, which rearrange the heavy and light chain genes responsible for producing clonally diverse immunoglobulin (Ig) molecules. These cells then differentiate into immature B cells, followed by mature B cells. The bone marrow is a complex ecological niche of supporting stromal cells, extracellular matrix components, macrophages, and hematopoietic precursor cells influencing B-cell development, maturation, and differentiation. Once fully mature, B cells circulate in peripheral lymphoid organs and can respond to antigenic stimuli. As specific cell surface markers are expressed during each stage of B-cell development, researchers use flow cytometry as a powerful tool to evaluate developmental progression. In this protocol, we provide a step-by-step method for bone marrow isolation, cell staining, and data analysis. This tool will help researchers gain a deeper understanding of the progression of B-cell development and provide a pertinent flow gating strategy.

Keywords: B-cell development; Adaptive immunity; Bone marrow isolation; Flow cytometry; Lymphocyte staining.

©Copyright : © 2023 The Authors; This is an open access article under the CC BY license.

PubMed Disclaimer

Conflict of interest statement

Competing interestsThe authors report no competing interests.

Figures

Figure 1.

Figure 1.. Diagram of B-cell development.

Major stages of adult B-cell development in the bone marrow focusing on the cell surface markers relevant to this protocol.

Figure 2.

Figure 2.. Mouse dissection diagram focusing on the location and isolation of intact femur

Figure 3.

Figure 3.. Representative flow cytometry graphs post-compensation using the APC-Cy7-CD43 staining.

Isolated bone marrow B cells were stained with APC-Cy7-CD43 antibody following the protocol, to determine if it is detected by other channels being used (FITC, PE, BV-421, and PE); every channel is checked separately. This confirms the APC-Cy7-CD43 positive (69.5%) population is being identified in the appropriate channel, while compensation in other single channels eliminates/strongly reduces the bleed-through signal from overlap in spectral emission.

Figure 4.

Figure 4.. Representative flow cytometry plots for each stage of the gating pipeline for data analysis.

Sample is from a wildtype mouse.

References

    1. LeBien T. W. and Tedder T. F. B lymphocytes: how they develop and function (2008). Blood 112(5): 1570–1580. doi: 10.1182/blood-2008-02-078071 -DOI -PMC -PubMed
    1. Pieper K., Grimbacher B. and Eibel H. B-cell biology and development (2013). J. Allergy Clin. Immunol. 131(4): 959–971. doi: 10.1016/j.jaci.2013.01.046 -DOI -PubMed
    1. Wells S. M., Kantor A. B. and Stall A. M.(1994). CD43(S7) expression identifies peripheral B cell subsets. J. Immunol. 153(12): 5503–5515.. doi: 10.4049/jimmunol.153.12.5503 -DOI -PubMed
    1. Perez-Andres M., Grosserichter-Wagener C., Teodosio C., van Dongen J. J., Orfao A. and van Zelm M. C.(2011). The nature of circulating CD27+CD43+ B cells. J. Exp. Med. 208(13): 2565–2566.. doi: 10.1084/jem.20112203 -DOI -PMC -PubMed
    1. 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(5): 1213–1225.. doi: 10.1084/jem.173.5.1213 -DOI -PMC -PubMed

Grants and funding

LinkOut - more resources