2.15. Mitochondrial H2O2 Measurement

H₂O₂ emission was determined by the fluorogenic indicator Amplex Red (Invitrogen) oxidation in presence of horseradish peroxidase as described by Starkov [44]. Fluorescence was recorded in a spectrofluorometer (LS 55 PerkinElmer Life Sciences) with excitation and emission wavelengths of 555 and 581, respectively. Briefly, 300 µg of purified mitochondria was added to 1 mL incubation buffer containing 125 mM KCl, 20 mM Hepes, 0.2 mM EGTA, 2 mM KH₂PO₄, 2% BSA, 1 µM Amplex Red, and 4 U horseradish peroxidase (pH 7.2). H₂O₂ production was initiated after addition of 5 mM pyruvate, 2.5 mM malate, and 10 mM succinate as substrates and 1 µM rotenone, 0.2 µM antimycin A, and 5 mM malonate as inhibitors.