Methods to measure cytoplasmic and mitochondrial Ca(2+) concentration using Ca(2+)-sensitive dyes

Ca(2+) is a ubiquitous second messenger that is involved in regulation of various signaling pathways. Cytoplasmic Ca(2+) is maintained at low concentrations (~100 nM) by many active mechanisms. Increases in intracellular Ca(2+) concentration ([Ca(2+)]i) indeed can initiate multiple signaling pathways, depending both on their pattern and subcellular localization. In T cells, the stimulation of T-cell receptor leads to an increase in [Ca(2+)]i upon the opening of Ca(2+) release-activated calcium (CRAC) channels. T cells can actually sustain high [Ca(2+)]i for several hours, resulting in the activation of transcriptional programs orchestrated by members of the nuclear factor of activated T-cell (NFAT) protein family. Here, we describe an imaging method widely employed to measure cytoplasmic [Ca(2+)] in naïve and effector T cells based on the ratiometric dye Fura-2. Furthermore, we discuss a pharmacological method relying on an inhibitor of CRAC channels, 2-aminoethyldiphenyl borate, to validate the role of CRAC channels in cytoplasmic Ca(2+) elevation. Finally, we describe an approach to measure mitochondrial [Ca(2+)] based on another fluorescent dye, Rhod-2. With appropriate variations, our methodological approach can be employed to assess the effect and regulation of cytosolic and mitochondrial Ca(2+) waves in multiple experimental settings, including cultured cancer cells.