Oxygen consumption rate (OCR) measurements using Seahorse XF Cell Mito Stress Test

HepG2 cells were seeded in XF24 Cell Culture Microplate in normal growth medium. 24 h prior to the assay, growth medium was replaced with substrate-limited medium containing 0.5 mM glucose, 1 mM GlutaMAX, 0.5 mM carnitine, and 1% FBS. 45 min prior to the assay, the cells were washed for two times with FAO Assay Medium containing 111 mM NaCl, 4.7 mM KCl, 1.25 mM CaCl₂, 2 mM MgSO₄, 1.2 mM NaH₂PO₄, 2.5 mM glucose, 0.5 mM carnitine, and 5 mM HEPES adjusted to pH 7.4 at 37 °C. 375 µL/well FAO assay medium was added to the cells and cells were further incubated in a non-CO₂ incubator for 30 min at 37 °C. Meanwhile, the assay cartridge was loaded with XF Cell Mito Stress Test compounds (final concentrations: 2.5 µg/mL oligomycin, 2 µM FCCP, 2 µM rotenone, and 4 µM antimycin A). 15 min prior to starting the assay, cells were incubated in Eto-containing FAO Assay Medium (final concentration: 40 μM) to allow for efficient inhibition of CPT1. After 15 min incubation at 37 °C in a non-CO2 incubator, the XF Cell Culture Microplate was inserted into the XF24 Analyzer and run the XF Cell Mito Stress Test according to the manufacturer’s instructions.