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

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 F₀F₁-ATP synthase (F₀F₁), respiratory chain, TCA cycle and ATP/ADP translocator, as well as knockdown of β-subunit of F₀F₁, 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.