Mitochondrial Hydrogen Peroxide Quantification

Approximately 50 mg of liver tissue was homogenized in 1 ml of homogenization buffer containing 25 mM Hepes pH7.4, 1 mM EDTA, 0.25 M sucrose, 2 mM MgCl₂, 1 μM butylated hydroxytoluene (BHT), 1:200 dilution of Sigma P8340 and 1 μM diethylenetriaminepentaacetic acid (Sigma-Aldrich). The homogenates were centrifuged at 500 × g for 5 min to pellet nuclei and debri. The resultant supernatant was centrifuged at 10,000 × g for 10 min at 4 °C to obtain mitochondria. The pellets were resuspended in 150 μl homogenization buffer. About 20 μL solution was saved for measuring protein concentration.
Mitochondrial H₂O₂ was detected using Amplex Red assay as described39 (link). The working solution was prepared using 100 μL of 10 mM Amplex Red reagent (Thermo Fisher Scientific), 2 μL of 1000 U/ml horseradish peroxidase (HRP) and 10 ml of 50 mM potassium phosphate (pH7.7) with 0.5 mM diethylenetriaminepentaacetic acid (Sigma-Aldrich). 50 μL of sample or standard was pipetted into 96 plates, followed by addition of 50 μl of the Amplex Red reagent/HPR working solution. The reaction mixture was protected from light and incubated at room temperature for 30 min. The fluorescence was measured with excitation in the range of 530–560 nm and emission at 590 nm. Mitochondrial H₂O₂ levels were normalized to protein levels.