4.9. Measurement of Mitochondrial ROS Production

ROS production by mitochondria was measured using Amplex red in a Biotek Cytation 3 reader at 560/590 nm excitation and an emission wavelength, respectively. The assay was performed in the presence of 100 µg of freshly isolated mitochondria in a reaction buffer containing 50 mM Tris-HCl, pH 7.4, 75 mM sucrose, 225 mM mannitol, 5 µM Amplex Red, 40 U/mL superoxide dismutase (SOD), and 20 U/mL horseradish peroxidase, supplemented with 10 mM succinate and 2 µM rotenone.