T细胞钙离子流动性分析(流式细胞仪)

Guo N. Huang

doi:10.21769/BioProtoc.171

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Peer-reviewed

T Cell Calcium Mobilization Study (Flow Cytometry)

T细胞钙离子流动性分析(流式细胞仪)

GH Guo N. Huang email

发布: 2012年05月05日第2卷第9期 DOI: 10.21769/BioProtoc.171 浏览次数: 16150

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Abstract

Antigen recognition and activation of T cell receptor (TCR) triggers transient calcium release from intracellular compartments and subsequent sustained calcium influx through cell surface Icrac channels. Sustained elevation of the cytoplasmic calcium level activates many calcium-dependent enzymes and transcription factors, which are essential for T cell activation and function. This protocol uses non-ratiometric dyes, in combination with flow cytometry, to monitor TCR-triggered calcium changes over time, and is a simple assay to examine the existence of T cell calcium mobilization defects in transgenic mice.

Materials and Reagents

Anti-CD3 Armernian hamster primary antibody (BD Biosciences, catalog number: 553057 , NA/LE)
Goat anti-Armernian Hamster IgG antibody (Jackson ImmunoResearch, catalog number: 127-005-099 )
Phosphate buffered saline (PBS)
CaCl₂
MgCl₂
DMSO (Merck KGaA, Calbiochem^®, catalog number: 540025 )
Fluo-3 AM in DMSO (Life Technologies, Molecular Probes^®, catalog number: F-1242 )
(Note: Fluo-3 fluorescence is calcium-dependent) (see Recipes)
Fura Red AM in DMSO (Life Technologies, Molecular Probes^®, catalog number: F-3020 ) (Note: Fura Red fluorescence is calcium-independent. Fura Red serves as a control of dye loading efficiency) (see Recipes)
Pluronic F-127 in DMSO (Life Technologies, Molecular Probes^®, catalog number: F-1242) (see Recipes)
Hanks buffered solution (HBSS) (Life Technologies, Gibco^®, catalog number: 14170-112 ) (see Recipes)
Dye loading buffer (see Recipes)