Assay of mitochondrial respiratory function. Several parameters of mitochondrial respiration are derived by measuring the oxygen consumption rate (OCR) upon the sequential addition of mitochondrial inhibitors. Basal OCR is first measured, from which nonmitochondrial respiration is subtracted to derive basal respiration. Oligomycin, a complex V inhibitor, is used to derive ATP-linked respiration (basal OCR minus oligomycin OCR) and proton leak respiration (oligomycin OCR minus nonmitochondrial respiration). Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), a protonophore, collapses the inner membrane proton gradient, allowing the ETC to function at its maximal rate, and maximal respiratory capacity can be derived (FCCP OCR minus nonmitochondrial respiration). Antimycin A and rotenone, inhibitors of complexes III and I, inhibit all ETC function, revealing the nonmitochondrial respiration. Reserve capacity is derived from the maximal and basal rates (maximal OCR minus basal OCR).