Calcium ions (Ca2+) are key second messengers for signal transduction in virtually all cells. In T cells, Ca2+ signals are generated upon T cell receptor (TCR) stimulation in a two-step integrated process known as Store-Operated Ca2+ Entry (SOCE), which involves the depletion of endoplasmic reticulum (ER) Ca2+ stores, followed by the influx of extracellular Ca2+ via Ca2+ release-activated Ca2+ (CRAC) channels. The Ca2+ influx generated by the opening of CRAC channels in T cells is essential for their metabolic reprogramming, proliferation, cytokine production, and adaptive immune response. In this book chapter, we review general concepts, discuss the rationale for using ratiometric Ca2+-sensitive chemical dyes to monitor SOCE in primary murine T cells, and weigh the advantages and disadvantages of the different methods that are currently available to detect cytosolic Ca2+ dynamics. We provide detailed protocols to measure SOCE in mouse T cells including flow cytometry, fluorescent microplate reader and single-cell imaging, and offer a general guideline on how to quantify SOCE in these cells. These protocols are easily adaptable to monitor cytosolic Ca2+ dynamics in human T cells and other cell types of interest.

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Analysis of Store-Operated Ca2+ Entry in Primary T Cells

  • Sara T. Granados,
  • Sergei Yanushkevich,
  • Jessica Lok,
  • Axel R. Concepcion

摘要

Calcium ions (Ca2+) are key second messengers for signal transduction in virtually all cells. In T cells, Ca2+ signals are generated upon T cell receptor (TCR) stimulation in a two-step integrated process known as Store-Operated Ca2+ Entry (SOCE), which involves the depletion of endoplasmic reticulum (ER) Ca2+ stores, followed by the influx of extracellular Ca2+ via Ca2+ release-activated Ca2+ (CRAC) channels. The Ca2+ influx generated by the opening of CRAC channels in T cells is essential for their metabolic reprogramming, proliferation, cytokine production, and adaptive immune response. In this book chapter, we review general concepts, discuss the rationale for using ratiometric Ca2+-sensitive chemical dyes to monitor SOCE in primary murine T cells, and weigh the advantages and disadvantages of the different methods that are currently available to detect cytosolic Ca2+ dynamics. We provide detailed protocols to measure SOCE in mouse T cells including flow cytometry, fluorescent microplate reader and single-cell imaging, and offer a general guideline on how to quantify SOCE in these cells. These protocols are easily adaptable to monitor cytosolic Ca2+ dynamics in human T cells and other cell types of interest.