Measurement of extracellular acidification rate (ECAR) and oxygen consumption rate (OCR)

The Seahorse Bioscience XF96 Extracellular Flux Analyzer (Seahorse Bioscience, USA) was used to monitor mitochondrial function and glycolytic capacity with Seahorse XF Cell Mito Stress Test Kit or Glycolysis Stress Test Kit (Seahorse Bioscience, Billerica, MA, USA). In brief, cells were seeded in a XF96-well plate at a density of 4 × 10⁴ per well and allowed to attach overnight. For ECAR assessment, cells were incubated with unbuffered medium followed by a sequential injection of 10 mM glucose, 1 μM oligomycin, and 80 mM 2-DG. The mitochondrial respiration (OCR) was measured by sequential injection of 1 μM oligomycin, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) and 2 μM antimycin A and rotenone. The ECAR and OCR value were normalized to the total protein content between groups prior to making comparisons.