Analysis of in vitro mitochondrial respiration

Analysis of mitochondrial respiratory potential was performed using a flux analyzer (Seahorse XF^e96 Extracellular Flux Analyzer; Seahorse Bioscience, North Billerica, MA, USA) with a Seahorse XF Cell Mito Stress Test Kit according to the manufacturer’s instructions. Basal respiration and ATP production were calculated to evaluate mitochondrial respiratory function according to the manufacturer’s instructions. After the measurement, cells were harvested to count the cell number, and each plotted value was normalized relative to the number of cells used. Briefly, NPCs were seeded (25,000 cells/well) into each well of XF^e96 cell culture plates and were maintained in standard culture media. After 2–3 days in culture, cells were equilibrated in unbuffered XF^eassay medium (Seahorse Bioscience) supplemented with glucose (4.5 g/L), sodium pyruvate (25 mg/L) and transferred to a non-CO₂ incubator for 1 hr before measurement. Oxygen consumption rate (OCR) was measured with sequential injections of oligomycin, FCCP, and rotenone/antimycin A.