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

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Author: Antony, P
Author: Khan, WN
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Methods Article

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

Pierre Antony¹, Kristen Hoek¹, Bhaskarjyoti Sarmah¹ and Wasif N. Khan¹^*

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.

Figure 4 Enlarged

Fig. 4:

Calcium mobilization in B cell subsets. (A): MACS-enriched B cells were loaded with Indo-1-AM (5 μg/ml), and labeled with AA4.1, anti-CD23, and anti-HSA for B cell subset identification. (B): Cells were applied to a cytometer, basal Ca²⁺ levels were monitored for 90 s, then cells were stimulated with 10 μg/ml anti-IgM (indicated by the arrow) and monitored for 4-6 min; T1 (dotted black line), T2 (black line), mature FoB (gray line), MZ (dotted gray line). Data is a representative of 4-6 independent experiments.

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