H2O2 production in mitochondria was measured using the Amplex Ultra Red fluorescent reagent (Invitrogen, Carlsbad, CA, USA) according to the protocol described earlier [76 (link)]. The Amplex Red assay is a most specific and sensitive method, with a limit of detection less than 5 pmol of H2O2. The stoichiometry of Amplex Red and H2O2 is 1:1, and, therefore, the assay results are linear over the range of values encountered in tissues and cells [76 (link)]. The excitation wavelength was set to 530 nm and the emission wavelength to 590 nm. The measurements were carried out using a Hitachi F-7000 fluorescence spectrophotometer (Hitachi High Technologies, Tokyo, Japan).
The substrate (10 mM pyruvate), 4 mM phosphate (KH2PO4), 1 U of Amplex Ultra Red reagent, 4 U of horseradish peroxidase (Amresco, Solon, OH, USA), and 0.2 mg of mitochondria were added to 1 mL of isolation buffer (225 mM mannitol, 75 mM sucrose, 5 mM Hepes (pH 7.4), 1 mM EGTA, 2 mg/mL fatty acids free BSA. The H2O2 concentration was measured as the fluorescence intensity of resorufin formed during the reaction upon oxidation of Amplex Ultra Red. Production changes were recorded after the addition of 0.2 mg cisplatin, 1 μM methylene blue, and 1 μM azure B.
The substrate (10 mM pyruvate), 4 mM phosphate (KH2PO4), 1 U of Amplex Ultra Red reagent, 4 U of horseradish peroxidase (Amresco, Solon, OH, USA), and 0.2 mg of mitochondria were added to 1 mL of isolation buffer (225 mM mannitol, 75 mM sucrose, 5 mM Hepes (pH 7.4), 1 mM EGTA, 2 mg/mL fatty acids free BSA. The H2O2 concentration was measured as the fluorescence intensity of resorufin formed during the reaction upon oxidation of Amplex Ultra Red. Production changes were recorded after the addition of 0.2 mg cisplatin, 1 μM methylene blue, and 1 μM azure B.