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

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.