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

被引:3
作者
Antony, Pierre [1 ]
Hoek, Kristen [1 ]
Sarmah, Bhaskarjyoti [2 ]
Khan, Wasif N. [1 ]
机构
[1] Vanderbilt Univ, Sch Med, Dept Microbiol & Immunol, Nashville, TN 37232 USA
[2] Vanderbilt Univ, Sch Med, Dept Cell & Dev Biol, Nashville, TN 37232 USA
关键词
receptors; antigen; b-cell; immunomagnetic separation;
D O I
10.1251/bpo135
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 x 10(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 1 x 10(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.
引用
收藏
页码:73 / 83
页数:11
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