Xfp analyzer system

Cells were seeded in Seahorse XFp cell culture microplates at 8 × 10³ cells/well for 24 h and then treated with ouabain as indicated for another 24 h. The culture medium was replaced with seahorse assay medium, and cells were incubated at 37 °C in a CO₂-free incubator for 1 h. The assay medium was prepared by adding 100 mM sodium pyruvate, 200 mM L-glutamine and 2.5 M glucose to the Agilent Seahorse XF Base Medium. The seahorse assay was run in an XFp Analyzer System (Agilent Technologies) following the manual provided by the manufacturer. To assess mitochondrial metabolic function, oligomycin (1.0 μM), carbonyl cyanide-p-(trifluoromethoxy) phenylhydrazone (FCCP, 1.0 μM), antimycin, and rotenone (0.5 μM) were sequentially injected, and the measurements were recorded after each injection. After measuring the baseline oxygen consumption rate (OCR), the reduction in OCR following oligomycin injection correlates with mitochondrial respiration associated with cellular ATP production. The FCCP-stimulated OCR was used to measure spare respiratory capacity, i.e., the difference between maximal respiration and basal respiration. Rotenone and antimycin A were used to inhibit electron transfer chain (ETC) complexes I and III, respectively, to shut down mitochondrial respiration.

Cells were seeded in Seahorse XFp cell culture microplates and treated with ouabain as stated above. The assay medium was prepared by adding 200 mM L-glutamine to the Agilent Seahorse XF Base Medium. The seahorse assay was run in an XFp Analyzer System (Agilent Technologies) following the manufacturer’s instructions. To assess the glycolytic function, glucose (10 mM), oligomycin (1.0 μM), and 2-deoxyglucose (2-DG, 50 μM) were sequentially injected, and the measurements were recorded after each injection. After measuring the extracellular acidification rate (ECAR), the increase in ECAR following glucose correlates with the rate of glycolysis under basal conditions. Oligomycin was used to inhibit mitochondrial ATP production and shift energy production to glycolysis. The increase in ECAR following oligomycin reveals the maximal cellular glycolytic capacity. 2-DG was used to inhibit glycolysis through competitive binding to glucose hexokinase. The decrease in ECAR following 2-DG confirms that the ECAR produced is due to glycolysis.