Seahorse xf96 extracellular flux analyzer

OCR was assessed in real-time with a Seahorse XF96 Extracellular Flux Analyzer (Seahorse Bioscience, Billerica, MA, USA), which allows to measure OCR changes after sequential addition of modulators of respiration that target components of the electron transport chain in mitochondria. Cells (1 × 10⁴ cells/well/200 µL of DMEM) were plated in a XF 96 cell culture microplate (Seahorse Bioscience Inc., Billerica, MA, USA). Cells were washed with base media once, immersed in 175 µL base media, and incubated in the absence of CO₂ for 20 min. After baseline measurements, respiration was measured after sequentially adding 25 µL of oligomycin (inhibitor of ATP synthase, 1 µg/mL), carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) (a protonophore and uncoupler of mitochondrial oxidative phosphorylation, 0.5 μM), and a combination of rotenone (mitochondrial complex I inhibitor, 1 μM) and antimycin A (mitochondrial complex III inhibitor, 1 μM) for OCR measurement using the XF Cell Mito Stress Test Kit (Cat. No. 103015-100, Seahorse Bioscience Inc., Billerica, MA, USA). OCR values were normalized for the protein content of each sample and expressed as the unit of pmoles/min. Basal OCR was expressed as percentage of the untreated cells (None). Basal OCR for “None” was set at 100.

The intact cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured in real time using the Seahorse XF96 Extracellular Flux Analyzer (Seahorse Bioscience). Briefly, 1.0 × 10⁴ H1299 or 2.0 × 10⁴ HepG2 cells were seeded into 96-well Seahorse microplates in 80 μL of growth medium and incubated at 37 °C in 5% CO₂ for 16 h and the calibrator plate was equilibrated overnight in a non-CO₂ incubator. Before starting the test, cells were washed twice with assay running media (unbuffered DMEM, 25 mmol/L glucose, 1 mmol/L glutamine, 1 mmol/L sodium pyruvate for OCR; unbuffered DMEM, 1 mmol/L glutamine for ECAR) and equilibrated in a non-CO₂ incubator. Once the probe calibration was completed, the probe plate was replaced by the cell plate. The protocol was optimized for the simultaneous measurement of OCR and ECAR. For OCR, the analyzer plotted the value as the cells were treated by sequential injection of the following compounds: oligomycin (1 μmol/L), carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP, 0.5 μmol/L), and antimycin A (1 μmol/L) plus rotenone (1 μmol/L). For ECAR, the analyzer plotted the value as the cells were treated by sequential injection of the following compounds: glucose (10 mmol/L), oligomycin (1 μmol/L) and 2-deoxy-glucose (2-DG, 100 mmol/L).

S-MSCs were seeded at a density of 4 × 10⁴ cells per well in XF 96-well plates. After induction of differentiation into BAs, the OCR was measured with a Seahorse XF96 Extracellular Flux Analyzer (Seahorse Bioscience, Billerica, MA, USA) following the manufacturer’s instructions. Oligomycin and rotenone were used as inhibitors, and the uncoupler FCCP was used as an agonist. The results were calculated and are presented as basal respiration, ATP production, proton leakage, maximal respiration, and spare capacity.