Measurement of mitochondrial respiration

Cells were seeded at 4.5 × 10⁴ cells/well in 200 μl growth medium in XF 24-well cell culture microplates (Seahorse Bioscience) and incubated at 37°C in 5% CO2 for 24 hr. One hour before the assay, growth medium was removed and replaced with assay medium (low buffered DMEM, 10 mM l-glutamine, 1 mM sodium pyruvate, 5 mM galactose), with one wash of assay medium, and left to stabilise in a 37°C non-CO₂ incubator. Analysis was performed in quadruplicate using a XF24 Extracellular Flux Analyzer (Seahorse Bioscience). The wells were sequentially injected with 100 nM oligomycin to inhibit ATP synthase, 700 nM carbonylcyanide-4-trifluorometho-xyphenylhydrazone (FCCP) to uncouple the respiratory chain, and 200 nM rotenone to inhibit complex I. Oxygen consumption rate (OCR) was measured for each well every 5 min before and after each injection.