title: Micro-scale flow cytometry-based and biochemical analysis of lipid signaling in primary B cell subpopulations.
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 0.5x10(6) 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 1x10(6 )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.
authors.affiliation: Vanderbilt University School of Medicine, USA.
authors.forename: Pierre
authors.initials: P
authors.lastname: Antony
authors.forename: Kristen
authors.initials: K
authors.lastname: Hoek
authors.forename: Bhaskarjyoti
authors.initials: B
authors.lastname: Sarmah
authors.forename: Wasif N
authors.initials: WN
authors.lastname: Khan