Mitochondrial ROS level quantification

The mitochondrial ROS measurement was performed through flow cytometry by using DCFH-DA (2,7-dichlorodi-hydrofluorescein diacetate). Isolated liver mitochondria (0.5 mg protein per mL) were incubated with Pb²⁺ in respiration buffer MRB (2 mL, containing 0.32 mM sucrose, 10 mM Tris, 20 mM Mops, 50 mM EGTA, 0.5 mM MgCl₂, 0.1 mM KH₂PO₄ and 5 mM sodium succinate, pH 7.4) for 15 min at 25 °C. The samples with DCFH-DA (5 μM) were incubated for 10 min and then determined through flow cytometry (BD, C6) equipped with a 488 nm argon ion laser and supplied with the Cytometer software. The signals were obtained using a 530 nm bandpass filter (FL-1 channel). Each determination is based on the mean fluorescence intensity of 50 000 counts.15