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


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.



B cell antigen receptor




Dulbecco’s modified Eagle’s media

IP3 :

inositol triphosphate


flow cytometry


magnetic bead cell sorting


mean fluorescence intensity


protein kinase C


red blood cell


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Correspondence to Wasif N. Khan PhD..

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These authors contributed equally.

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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).

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Indexing terms

  • receptors, antigen, b-cell
  • immunomagnetic separation