Multiskan microtiter plate reader

The dihydroorotate dehydrogenase activity assay protocol was similar to that used for succinate dehydrogenase activity using 10 mM dihydroorotate (DOA) instead of succinate. The NBT reduction evoked by the superoxide generation mediated by dihydroorotate dehydrogenase was measured every 5 min spectrophotometrically at 595 nm in a Multiskan microtiter plate reader (Thermo Scientific).

The NBT reduction evoked by the superoxide generation mediated by glycerol 3‐phosphate dehydrogenase was measured using the succinate dehydrogenase assay medium with 10 mM glycerol 3‐phosphate (G3P) instead of succinate. Measures were acquired every 5 min spectrophotometrically at 595 nm in a Multiskan microtiter plate reader (Thermo Scientific).

The superoxide production evoked by the activity of the mitochondrial complex II, Succinate dehydrogenase. The assay buffer consisted of a 50 mM phosphate buffer (Na₂HPO₄, NaH₂PO₄, pH 7.4), 0.5 mg/ml nitro blue tetrazolium (NBT) as a redox dye and 1 mM succinate as a complex II substrate. The reaction was started by the addition of 0.1 mg/ml membranes homogenates for 360 min (25°C) with or without 50 μM dUQ. The NBT reduction evoked by the superoxide generation mediated by CII activity was measured every 5 min spectrophotometrically at 595 nm in a Multiskan microtiter plate reader (Thermo Scientific).