Oxygen consumption rate measurement

Oligomycin, an inhibitor of ATP synthase, was prepared from 1000× stock at a concentration of 10 mM in dimethyl sulfoxide (DMSO). FCCP, an ionophore and strong mitochondrial depolarizer, was prepared from 1,000 × stock at a concentration of 5 mM in DMSO. Rotenone, a potent inhibitor of mitochondrial complex I, and antimycin A, a strong suppressor of mitochondrial complex III, were solubilized from 1,000 × stock solutions at concentrations of 10 mM in DMSO. To measure SCLC-cell oxygen consumption rates (OCRs), 6 × 10⁴ cells from each cell line were seeded into each well of an XF96 microplate 16 hours before the experiment. Immediately before the OCR measurement, culture medium of the cells was replaced by an assay medium (low-buffered RPMI containing 25 mM D-glucose, 1 mM sodium pyruvate, and 1 mM L-glutamine) and incubated for 1 hour at 37°C. The OCR in the cells were measured using an Extracellular Flux Analyser (Seahorse Biosciences). After baseline measurements of OCR the inhibitors described above prepared in the assay medium were sequentially injected into each well to reach the final working 1× concentrations. After 5 minutes of incubation to expose cancer cells equally to chemical inhibitors, the OCR was measured again. Data were analyzed using the Seahorse XF software program. The OCR was reported in pmol min−1, and measurements were normalized according to the final cell number.