2.6. Seahorse extracellular flux analysis of mitochondrial respiration

Agilent Seahorse XF Cell Mito Stress Test was applied to HepG2^PRDX6+/+ and HepG2^PRDX6-/- cells and oxygen consumption rate (OCR) determined using Agilent Seahorse XF24 Analyzer (Agilent Seahorse Bioscience, Santa Clara, CA, USA). 48 h before the assay, cells were seeded at 40,000 cells per well in a Seahorse 24-well XF Cell Culture microplate in 250 μL of culture medium and were allowed to adhere for 24 h in 5% CO₂ atmosphere at a 37 °C. In addition, the Seahorse XF Sensor Cartridge was hydrated for around 24 h with 500 μL of Seahorse XF Calibrant Solution in a non-CO₂ atmosphere at 37 °C to remove CO₂ from the media that would interfere with measurements. After that, cells were washed three times with XF Assay Medium supplemented with 10 mM glucose, 2 mM sodium pyruvate and 2 mM glutamine, pH 7.4, and then maintained in XF assay media at 37 °C in a non-CO₂ incubator for 1 h. Mitochondrial function of the cells was analyzed by sequential injections of the modulators oligomycin (1 μM), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, 1 μM) and rotenone (0.5 μM), dissolved in pre-warmed XF Assay Medium and loaded into the designated injection ports of the hydrated sensor cartridge. The loaded XF Sensor Cartridge with a XF Utility Plate was placed into the XF24 Analyzer and calibrated. After calibration, the XF Utility Plate with the calibration fluid was replaced with the plate containing cells. Measurement cycles were performed at the beginning and after addition of each modulator and the parameters basal, ATP production-linked, maximal, proton leak-linked OCR, spare respiratory capacity and non-mitochondrial respiration determined following manufacturer's guidelines. Data were normalized to protein concentration determined at the end of the assay.