Measurement of oxygen consumption rate (OCR) in cells

OCR was measured using the Seahorse Extracellular Flux (XF24) Analyzer (Agilent Technologies, Santa Clara, CA). Seahorse assay media (DMEM without glucose, L-glutamine, phenol red, sodium pyruvate, and sodium bicarbonate [Sigma-Aldrich, St. Louis, MO] prepared with 6 mM glucose, 1.85 g/l sodium chloride, 1 mM sodium pyruvate, and 15 mg/l phenol red) was freshly supplemented with 2 mM L-glutamine and the pH adjusted to 7.35 with sodium hydroxide. BET1A cells were detached, washed 3 times with Seahorse assay medium, and resuspended in assay medium. 6×10⁵ cells in 150 μl assay medium were added to the wells of a Seahorse cell plate and adhered using BD Cell-Tak (BD Biosciences, Bedford, MA) according to Seahorse protocol for nonadherent cells, with 2 or 4 wells per plate left empty for background correction. The plate was incubated in a 37°C non-CO₂ incubator for 25 minutes. Additional assay medium was added to bring the final per-well volume to 500 μl and the plate incubated in a 37°C non-CO₂ incubator for an additional 15 minutes. The plate was then transferred to the Seahorse XF24 Analyzer for analysis. Once in the XF24, BET1A cells underwent a MitoStress test (basal measurement of oxygen consumption followed by successive treatments with oligomycin A [0.5 μM], FCCP [carbonyl cyanide-ρ-trifluoromethoxyphenylhydrazone; 0.5 μM], and rotenone and antimycin A [1.5 μM]). All MitoStress OCR measures were done 3 times in a 3-2-3-minute mix-wait-measure cycle.