Mitochondrial respiration analysis using the Seahorse XF Analyzer

To calculate the oxygen consumption rate (OCR), the Mito Stress Test Kit was used. The probe plate was hydrated with HPLC grade water in a CO₂-free incubator prior to metabolism calculation. In order to preserve the pH value, the test phenol red-free solution containing 10 mM glucose, 2 mM glutamine, 1 mM pyruvate and 5 mM HEPES was kept in a 37 °C CO₂-free incubator. In the hydration plate, the HPLC grade water was then replaced with a calibration solution and stored in a CO₂-free 37 °C incubator. When density of cells was at 5000 per well, HUVECs were seeded into XF96 cell culture microplates (Seahorse Bioscience) overnight and allowed to adherent to the tray. Then the cell culture medium was substituted with a red-free phenol assay solution and put for 1 h in a 37 °C CO₂-free incubator. Finally, the mitochondrial oxidative phosphorylation and glycolysis output rates of OCR were calculated and analyzed according to the manufacturer’s instructions and protocols on the Agilent Seahorse Bioscience XF96 Extracellular Flux Analyzer (Agilent Technologies) (Seahorse Bioscience, North Billerica, MA, USA).