利用流式细胞术分析T淋巴细胞中钙流变化

Sun-Hwa Lee

doi:10.21769/BioProtoc.910

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Peer-reviewed

Flow Cytometric Analysis of Calcium Influx Assay in T cells

利用流式细胞术分析T淋巴细胞中钙流变化

Sun-Hwa Lee email

发布: 2013年09月20日第3卷第18期 DOI: 10.21769/BioProtoc.910 浏览次数: 20476

评审: Anonymous reviewer(s)

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Abstract

Calcium influx is one of the key signaling events upon stimulation of T cell receptors (TCR) and plays an important role for T cell activation, proliferation, and differentiation. Phorbol myristate acetate (PMA) and calcium ionophore ionomycin are commonly utilized as stimulants in a variety of immunologic assays including T cell activation. PMA is a protein kinase C (PKC) activator, resulting in the activation of Ras, a small GPTase. When PMA and ionomycin are used together, TCR signaling downstream of PKC and Ras can be activated without activation of TCR-triggerd signaling events. This protocol describes the flow cytometry analysis of intracellular calcium influx in T cells stimulated with PMA and ionomycin.

Materials and Reagents

Jurkat T cells (ATCC, catalog number: TIB-152 )
DMSO (Sigma-Aldrich, catalog number: 472301 )
RPMI media 1640 (Life Technologies, Gibco^®, catalog number: 21875-034 )
Fetal Bovine Serum (FBS) (Life Technologies, Gibco^®, catalog number: 16000044 )
Penicillin/streptomycin (pen/strep) (Life Technologies, Gibco^®, catalog number: 15140-122 )
PMA (Sigma-Aldrich, catalog number: P1585 ) (Store at -20 °C)
Ionomycin (Sigma-Aldrich, catalog number: I0623 ) (Store at -20 °C)
Calcium Assay Kit (BD Biosciences, catalog number: 640176 )
Complete RPMI media (see Recipes)
PMA stock solution (see Recipes)
Ionomycin stock solution (see Recipes)