Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy.

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca) buffers to tightly regulate intracellular Ca concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (ΔΨm) to sequester Ca. The influx of Ca 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 Ca uptake and ΔΨm in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca can be measured using the fluorescent Ca 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 Ca and ΔΨm simultaneously. Using the sequential addition of Ca aliquots, mitochondrial Ca uptake can be monitored, and the concentration at which Ca induces mitochondrial membrane permeability transition and the loss of ΔΨm determined.