Bc1445

The activities of mitochondrial complexes I–V were monitored using detection kits (Cat# BC0515, BC3240, BC0945, BC1445, BC3235, Solarbio Life Sciences, Beijing, China). Briefly, approximately 0.1 g of brain tissue was taken from the injured area; mitochondrial extraction solution was used for mitochondrial extraction, and the extracted mitochondria were subjected to ultrasonic disruption (power 20%, ultrasonic for 5 seconds, interval of 10 seconds, 15 times). According to the functions of different mitochondrial complexes, the samples were added to the corresponding working solution, and the spectrophotometric value was measured after incubation for a certain time period; the activities of different mitochondrial complexes were calculated following the manufacturer’s instructions.

Mitochondrial membrane potential, ROS concentration, and activities of mitochondrial ETC I, II, and V were measured using commercially available kits (C2006 and S0033S, Beyotime, China; BC0515, BC3235, and BC1445, Solarbio, China), according to the manufacturer's instructions.

Complex (I, III, IV, and V) enzyme activity in cell extracts was measured by the single‐wavelength spectrophotometric assay using detection kits (cat. no. BC0510, BC3240, BC0945 and BC1445, Beijing Solarbio Science & Technology) according to the manufacturers’ protocols. Briefly, 5 × 10⁶ cells were collected and homogenized in lysis buffer. After two steps of centrifugation (600 × g for 10 min and then 11, 000 × g for 15 min) at 4˚C, the precipitate was collected for ultrasonic crushing and then added into the corresponding substrate to start the enzymatic reaction. The absorbance values at different wavelengths (I: 340 nm; III: 550 nm; IV: 550 nm; V: 660 nm) of the metabolic product was detected using a spectrophotometer (Thermo Fisher Scientific Inc.).
The OCR was measured using a 782 Oxygen Meter (Strathkelvin Instruments, Motherwell, UK) according to the manufacturer's instructions. The amplified signal was recorded at sampling intervals of 0.5 s.
Mitochondrial ATP was measured as previously described [34 (link)]. Briefly, the abundance of ATP was determined using the ATPlite assay system (PerkinElmer Inc., Waltham, MA, USA). Basal luminescence was recorded before the use of luciferin, and then cells were perfused with 20 μmol/L luciferin to record mitochondrial luminescence [35 (link)].