title: Methods to measure cytoplasmic and mitochondrial Ca(2+) concentration using Ca(2+)-sensitive dyes.
abstract: 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.
authors.affiliation: Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, California, USA. Electronic address: ssrikanth@mednet.ucla.edu.
authors.forename: Sonal
authors.initials: S
authors.lastname: Srikanth
authors.affiliation: Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.
authors.forename: Kyun-Do
authors.initials: KD
authors.lastname: Kim
authors.affiliation: Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.
authors.forename: Yousang
authors.initials: Y
authors.lastname: Gwack
mesh_terms: D000331:Aequorin / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002451:Cell Compartmentation; D045744:Cell Line, Tumor; D004396:Coloring Agents / Q000494:pharmacology*; D003593:Cytoplasm / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism
chemical_list: D004396:Coloring Agents; D000331:Aequorin; D002118:Calcium
keywords: CRAC channel; Fura-2; Orai; Rhod-2; STIM; Store-operated Ca(2+) entry; T cells