A sensitive, simple assay of mitochondrial ATP synthesis of cultured mammalian cells suitable for high-throughput analysis |
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Authors: | Makoto Fujikawa |
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Institution: | a ICORP ATP-Synthesis Regulation Project, Japan Science and Technology Agency (JST), Aomi 2-3-6, Tokyo 135-0064, Japan b Department of Molecular Bioscience, Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo-Motoyama, Kyoto 603-8555, Japan |
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Abstract: | 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 FoF1-ATP synthase (FoF1), respiratory chain, TCA cycle and ATP/ADP translocator, as well as knockdown of β-subunit of FoF1, 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. |
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Keywords: | FoF1-ATP synthase ATP synthesis Mitochondria High-throughput analysis Streptolysin |
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