Decreased glucose concentration depletes mitochondrial substrates causing a loss of inner membrane potential, and COX8-pHRed reported a decrease in matrix pH (red, n=17). Cytosolic pH reported by pHRed did not acidify when glucose is lowered (black, n=3). At the end of each experiment, consecutive 10 mM NH4Cl and 10 mM acetic acid pulses were used to verify that pHRed correctly reported an induced intracellular alkalinization and acidification, respectively. Note that the mitochondrial matrix (pH 8.0) rested alkaline relative to the cytosol (pH 7.3).