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
发布: 2013年09月20日第3卷第18期 DOI: 10.21769/BioProtoc.910 浏览次数: 20596
评审: Anonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Nov 2012
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)
Equipment
- Centrifuge (Eppendorf, model: 5810R )
- 37 °C 5% CO2 Cell culture incubator
- Cell Counter
- BD CantoII FACS machine
- 5 ml round-bottom FACS tube (BD Biosciences, catalog number: 352003 )
Software
- FACS DIVA software
- FlowJo software
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Lee, S. (2013). Flow Cytometric Analysis of Calcium Influx Assay in T cells. Bio-protocol 3(18): e910. DOI: 10.21769/BioProtoc.910.
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分类
免疫学 > 免疫细胞功能 > 淋巴细胞
细胞生物学 > 基于细胞的分析方法 > 离子分析
细胞生物学 > 基于细胞的分析方法 > 流式细胞术
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