4.15. Determination of the Oxygen Consumption Rate and Glycolysis Stress

The oxygen consumption rate (OCR) was measured using a Seahorse XFp Cell Mito Stress Assay (#103010-100, Agilent Technologies, Seoul, Korea) ran on a Seahorse XFp analyzer (Agilent Technologies, Seoul, Korea) according to the manufacturer’s instructions. Briefly, cells were seeded at 30,000 cells per well and treated with EE-84 for 48 h in 175 μL medium. Before measurements, plates were equilibrated in a CO₂-free incubator at 37 °C for 1 h. The analysis was performed using 1.5 µM oligomycin, 0.5 µM carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), and 1 µM rotenone/antimycin A as indicated. Data were analyzed using the Seahorse XF Cell Mito Stress Test report generator software (Agilent). The extracellular acidification rate (ECAR) was measured in response to the sequential injection of 10 mM glucose, 2 µM oligomycin (H+-ATP-synthase inhibitor), and 50 mM 2-deoxy-d-glucose (2DG) (hexokinase inhibitor) to detect non-glycolytic acidification, glycolysis, maximal glycolytic capacity, and glycolytic reserve using a Seahorse XFp analyzer with a Glycolysis Stress Test kit (Agilent, Santa Clara, CA, USA).