2.4 Mitochondrial H2O2 production

The H₂O₂ generated by actively respiring mitochondria was measured using Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (molecular probes, Cat#A22188) using a 96-well plate format. In a 130μl assay system, a series of MB concentrations from 0.0001 μg/ml to 1 μg/ml were prepared along with the combinations of mitochondrial substrates (Final concentrations 5 mM pyruvate, 2.5 mM malate and 150 μM ADP, 1 μM oligomycin A. 4 μM FCCP and 0.1 μM rotenone and 10 mM of succinate) prepared in Respiration buffer (RB mentioned in the OCR measurement protocol). The plate was placed in the 37°C pre-incubated plate reader (Biotek Synergy HT plate reader, Winooski, Vermont) and 25μl (5X) of Amplex solution (125μM Amplex + 0.5U/ml HRP) prepared in RB was added to each well using automated injectors. The fluorescence was measured at one-minute intervals at the wavelengths λ^EX 571 and λ^EM 585. The relative H₂O₂ generated was calculated from the difference in the fluorescence measurements.