Extracellular Flux Analysis of Hepatocyte Metabolism

Extracellular flux (XF) analysis deployed for real-time quantification of oxygen consumption rate (OCR) and the proton efflux rate (PER) with the Agilent Seahorse XF Cell Mito Stress Test kit and Agilent Seahorse XF Glycolytic Rate Assay kit, respectively. Both assays were formatted in Seahorse XF24 cell-culture microplates and performed as previously described [11 (link)]. HepG2 cells were seeded at a density of 2 × 10⁴ cells per well in a total of 200 µL for 24 h. Cells were serum-starved for 8 h and treated with vehicle control, 100 nM thapsigargin, or 1 µg/mL tunicamycin for 16 h prior to the initiation of measurements. To assess the effect of the pharmacological regulation of the intracellular H₂S levels, cells were co-treated with 100 µM NaHS, 100 µM HMPSNE, or 30 µM AOAA.