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.
Fujikawa
Makoto
M
ICORP ATP-Synthesis Regulation Project, Japan Science and Technology Agency (JST), Aomi 2-3-6, Tokyo 135-0064, Japan.
Yoshida
Masasuke
M
D000255:Adenosine Triphosphate / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D001426:Bacterial Proteins / Q000494:pharmacology; D002460:Cell Line; D002952:Citric Acid Cycle / Q000187:drug effects; D055785:Gene Knockdown Techniques; D057166:High-Throughput Screening Assays; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D010539:Permeability; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000235:genetics; D012680:Sensitivity and Specificity; D013301:Streptolysins / Q000494:pharmacology
D001426:Bacterial Proteins; D013301:Streptolysins; C025964:streptolysin O; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases