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Micro-scale flow cytometry-based and biochemical analysis of lipid signaling in primary B cell subpopulations

Biological Procedures Online volume 9, pages 73–83 (2007)Cite this article

Abstract

B cell subpopulations in the spleen have been extensively characterized phenotypically; however, biochemical properties of these cell populations following B cell antigen receptor engagement have not been fully determined due to technical difficulties and limiting cell numbers. We therefore employed mini-scale protocols to assess lipid signaling, particularly that of diacylglycerol and inositol trisphosphate, with as few as 0.5×106 purified early (T1) and late (T2) transitional B cells. Additionally, utilizing flow cytometric techniques, we determined levels of phosphatidylinositol bisphosphate and calcium mobilization in T1 and T2 cells, as well as mature follicular and marginal zone B cells using less than 1×106 primary B cells. Thus, these biochemical and flow cytometric methodologies can be used to analyse signal-induced changes in phosphatidylinositol bisphosphate levels, diacylglycerol and inositol triphosphate production and calcium in each B cell population.

Abbreviations

BCR:

B cell antigen receptor

DAG:

diacylglycerol

DMEM:

Dulbecco’s modified Eagle’s media

IP3 :

inositol triphosphate

FCM:

flow cytometry

MACS:

magnetic bead cell sorting

MFI:

mean fluorescence intensity

PKC:

protein kinase C

RBC:

red blood cell

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Authors and Affiliations

  1. Department of Microbiology and Immunology, Vanderbilt University School of Medicine, 37232-0146, Nashville, TN, USA

    Pierre Antony, Kristen Hoek & Wasif N. Khan PhD.

  2. Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, 37232-0146, Nashville, TN, USA

    Bhaskarjyoti Sarmah

Authors
  1. Pierre Antony
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  2. Kristen Hoek
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  3. Bhaskarjyoti Sarmah
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  4. Wasif N. Khan PhD.
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Corresponding author

Correspondence to Wasif N. Khan PhD..

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These authors contributed equally.

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Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Antony, P., Hoek, K., Sarmah, B. et al. Micro-scale flow cytometry-based and biochemical analysis of lipid signaling in primary B cell subpopulations. Biol. Proced. Online 9, 73–83 (2007). https://doi.org/10.1251/bpo135

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  • DOI: https://doi.org/10.1251/bpo135

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Biological Procedures Online

ISSN: 1480-9222

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