[Abstract]  [Full Text]  [PDF]

Pierre Antony¹, Kristen Hoek¹, Bhaskarjyoti Sarmah¹ and Wasif N. Khan^1*

¹ Vanderbilt University School of Medicine.

* To whom correspondence should be addressed: Wasif N. Khan, Vanderbilt University School of Medicine. Department of Microbiology and Immunology, Nashville, Tennessee 37232-0146.. USA. Phone: +615 343 5632. Fax: +615 343 7392. Email: wasif.khan@vanderbilt.edu

Biol. Proced. Online 2007;9:73-83. doi:10.1251/bpo135
Submitted: May 29, 2007; Accepted: December 09, 2007; Published: December 24, 2007.

Indexing terms: receptors, antigen, b-cell; immunomagnetic separation.

Abbreviations: BCR, B cell antigen receptor; DAG, diacylglycerol; DMEM, Dulbecco's modified Eagle's media; IP3, inositol triphosphate; FCM, flow cytometry; MACS, magnetic bead cell sorting; MFI, mean fluorescence intensity; PKC, protein kinase C; RBC, red blood cell.

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 0.5x10⁶ 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 1x10⁶ 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.