Synergy biotek

The mitochondrial CI activity was determined as reported elsewhere [52 (link)]. The tissue from an entire SN was homogenized in PBS pH 7.4 and subsequently centrifugated for 5 min at 2000 g. The supernatants were collected and used to perform the analyses. CI activity measurement is based on its ability to oxidize nicotinamide adenine dinucleotide + hydrogen (NADH) while reducing decylubiquinone (Dub) to dihydro-decylubiquinone (DUbH2), a molecule that is next oxidized by 2,6-Dichloroindophenol (DCPIP). This product can absorb light at 600 nm. In the meantime, 20 mM rotenone was included as a specific inhibitor of CI. All the measurements were completed employing a Synergy-Biotek (Biotek Instruments; Winooski, VT, USA) microplate reader at 37 °C. The CI activity was defined by subtracting the activity obtained with the inhibitor rotenone from the not inhibited activity and expressed as nmol/min/mg protein (data normalized by mean values of the not treated group).

Each tissue was homogenated in phosphate-buffered saline (PBS) pH 7.4, samples were centrifuged at 2000× g × 5 min, and the supernatants were used to carry out the determinations. Briefly, the measurement of CI activity was based on its capacity to oxidize nicotinamide adenine dinucleotide + hydrogen (NADH) while reducing decylubiquinone (Dub) to dihydro-decylubiquinone (DUbH2), which is then oxidized by 2,6-Dichloroindophenol (DCPIP). This oxidized product absorbs at 600 nm. Meanwhile, 20 mM rotenone was added as a specific inhibitor of CI. All the absorbance measurements were performed at 37 °C using a Synergy-Biotek (Biotek Instruments, Winooski, VT, USA) microplate reader. The specific activity of CI was determined by subtracting the activity in the presence of the inhibitor rotenone from the noninhibited activity and expressed as nmol/min/mg protein (these data were normalized by mean values of the untreated group).