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

Pierre Antony, Kristen Hoek, Bhaskarjyoti Sarmah, Wasif N. Khan

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)73-83
Number of pages11
JournalBiological Procedures Online
Volume9
Issue number1
DOIs
StatePublished - 2007
Externally publishedYes

Keywords

  • Antigen
  • B-cell
  • Immunomagnetic separation
  • Receptors

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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