Micro-scale flow cytometry-based and biochemical analysis of lipid signaling in primary B cell subpopulations |
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Authors: | Pierre Antony Kristen Hoek Bhaskarjyoti Sarmah Wasif N Khan |
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Institution: | (1) Department of Microbiology and Immunology, Vanderbilt University School of Medicine, 37232-0146 Nashville, TN, USA;(2) Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, 37232-0146 Nashville, TN, USA |
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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.
These authors contributed equally. |
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Keywords: | Indexing terms" target="_blank">Indexing terms receptors antigen b-cell immunomagnetic separation |
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