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

Abstract

<p class="first" id="d486455e120">Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca ²⁺) buffers to tightly regulate intracellular Ca ²⁺ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (ΔΨ _m) to sequester Ca ²⁺. The influx of Ca ²⁺ 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 ΔΨ _m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix. </p><p id="d486455e141">We describe here a method for simultaneously measuring mitochondria Ca ²⁺ uptake and ΔΨ _m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca ²⁺ can be measured using the fluorescent Ca ²⁺ indicator Fluo-4, AM, with measurement of ΔΨ _m 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 ²⁺ and ΔΨ _m simultaneously. Using the sequential addition of Ca ²⁺ aliquots, mitochondrial Ca ²⁺ uptake can be monitored, and the concentration at which Ca ²⁺ induces mitochondrial membrane permeability transition and the loss of ΔΨ _m determined. </p>