Mitochondrial stress test assay

Oxygen consumption rates were measured using a Seahorse Bioenergetic Flux analyzer (XFe96). Basal respiration and ATP-coupled respiration, represented as OCR, were measured using a Mitochondrial Stress Test assay as per the manufacturer’s instructions (Agilent, 103015-100). 20 000 cells per well were plated in a 96-well plate in RPMI 1640 medium (Corning) and 10% FBS (Gemini). Cells were incubated overnight at 37°C and 5% CO₂. The following day, Seahorse XF base medium (Agilent, 103193-100) was prepared by adding 2 mM Glutamine, and 10 mM glucose. No serum was present in XF base medium. The cells were washed twice with base medium and then, incubated at 37°C in a non-CO₂ incubator for 1 hour before the start of the assay. The concentrations of oligomycin, FCCP, Rotanone, and Antimycin A used for H23 and H1437 were 1 μM, 0.25 μM, 0.5 μM, and 0.5 μM, respectively. OCR values were normalized to DNA concentration per well as measured by CyQUANT (Thermo Fisher Scientific, C7026).