A sensitive, simple assay of mitochondrial ATP synthesis of cultured mammalian cells suitable for high-throughput analysis

► A sensitive, simple assay for mitochondrial ATP synthesis that represents mitochondrial functional integrity. ► It needs no detergent and no mitochondrial fractionation. ► Good for high-throughput analysis of mammalian cells cultured on a microplate. A new assay has been developed to measure mitochondrial ATP synthesis of cultured mammalian cells. Cells in a microplate are exposed to streptolysin O to make plasma membranes permeable without damaging mitochondrial function and ATP synthesis is monitored by luciferase. Addition of inhibitors of F oF 1-ATP synthase (F oF 1), respiratory chain, TCA cycle and ATP/ADP translocator, as well as knockdown of β-subunit of F oF 1, resulted in loss of ATP synthesis. Compared with the conventional procedures that need mitochondria fractionation and detergent, this assay is simple, sensitive and suitable for high-throughput analysis of genes and drugs that could affect mitochondrial functional integrity as represented by ATP synthesis activity.