title: Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy.
abstract: Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (ΔΨm) to sequester Ca2+. The influx of Ca2+into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains ΔΨm, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix. We describe here a method for simultaneously measuring mitochondria Ca2+uptake and ΔΨm in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+can be measured using the fluorescent Ca2+indicator Fluo-4, AM, with measurement of ΔΨm using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+and ΔΨm simultaneously. Using the sequential addition of Ca2+aliquots, mitochondrial Ca2+uptake can be monitored, and the concentration at which Ca2+induces mitochondrial membrane permeability transition and the loss of ΔΨm determined.
authors.affiliation: Centre for Genetic Diseases, Hudson Institute of Medical Research; The Department of Molecular and Translational Sciences, Monash University; matthew.mckenzie@hudson.org.au.
authors.forename: Matthew
authors.initials: M
authors.lastname: McKenzie
authors.affiliation: Centre for Genetic Diseases, Hudson Institute of Medical Research.
authors.forename: Sze C
authors.initials: SC
authors.lastname: Lim
authors.affiliation: Department of Physiology, University College London.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Duchen
mesh_terms: D000814:Aniline Compounds / Q000378:metabolism; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D002460:Cell Line; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods; D053078:Membrane Potential, Mitochondrial; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000379:methods*; D008928:Mitochondria / Q000378:metabolism*; D051336:Mitochondrial Membranes / Q000378:metabolism; D012235:Rhodamines / Q000378:metabolism; D014966:Xanthenes / Q000378:metabolism
chemical_list: D000814:Aniline Compounds; C409648:Fluo 4; D005456:Fluorescent Dyes; D012235:Rhodamines; D014966:Xanthenes; C005358:tetramethylrhodamine; D002118:Calcium