Mitochondrial ROS production was analyzed by using the Amplex Red H2O2 assay kit (Invitrogen) per manufacturer’s instruction. Mitochondria (250 μg) was added to the reaction mixture containing Amplex Red (50 μM), horse radish peroxidase (0.1 U/ml), superoxide dismutase (80 U/ml), and 5 mM glutamate / 2.5 mM malate or 5 mM succinate, and the fluorescence was measured at 590 nm with 540 nm excitation (APD130A2 Avalanche Photodetector, Thorlabs Inc.).
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