Seahorse extracellular flux (Seahorse XF) assays

Assays were performed using the Seahorse XFe96 analyzer (Seahorse Bioscience, Agilent) according to the manufacturer's instructions. The oxygen consumption rate (OCR) was measured using a Seahorse XF Cell Mito Stress Test Kit (Seahorse Bioscience, Agilent). Briefly, the sorted quiescent and cycling cells were seeded on Seahorse XF Cell Culture Microplate with assay medium (Seahorse XF Base Medium with 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose. pH = 7.4) for 1 h at 37 °C without CO₂. The OCR was measured under basal conditions and after the addition of oligomycin (1 μM), FCCP (1 μM), and rotenone (0.5 μM)/antimycin (0.5 μM). The extracellular acidification rate (ECAR) was measured using a Seahorse XF Glycolysis Stress Test Kit (Seahorse Bioscience, Agilent). Briefly, the isolated quiescent and cycling cells were seeded on Seahorse XF Cell Culture Microplate with assay medium (Seahorse XF Base Medium with 2 mM glutamine. pH = 7.4) for 1 h at 37 °C without CO₂. The ECAR was measured under basal conditions and after the addition of glucose (10 mM), oligomycin (1 μM), and 2-DG (50 mM).