Open Access

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

  • Pierre Antony1,
  • Kristen Hoek1,
  • Bhaskarjyoti Sarmah2 and
  • Wasif N. Khan1Email author
Biological Procedures Online9:91073

https://doi.org/10.1251/bpo135

Received: 29 May 2007

Accepted: 9 December 2007

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.

Indexing terms

receptors, antigen, b-cellimmunomagnetic separation

Notes

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