Calibration buffer

Minimum essential medium (MEM; cat. # 10-010-CV), MEM without phenol red (cat. # 17-305-CV), phosphate-buffered saline (PBS, cat. # 21-031-CV), glucose solution (cat. # 25-037-CI), and MEM nonessential amino acids (NEAA, cat. # 25-025-CI) were purchased from Corning, (New York, NY, USA). Dulbecco’s modified eagle medium without phenol red (DMEM, cat. # A14430-01), trypsin (cat. # 25300), penicillin-streptomycin solution (PenStrep, cat. # 15140-122), L-glutamine (L-Gln, cat. # 25030081), trypsin without phenol red (cat. # 15090046), and the BCA Protein Assay Kit (cat. # 23227) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The reagents 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; cat. # M2128), N, N-dimethylformamide (DMF; cat. # D4254), oligomycin (cat. # 75351), Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP; cat. # C2920), antimycin A (cat. # A8674), fetal bovine serum (FBS; cat. # F2442), and 2-deoxyglucose (2DG; cat. # D8375) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rotenone (cat. # 13995) was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Seahorse materials and reagents including 96-well cell culture plates and cartridges, calibration buffer, and XF Base media were purchased from Agilent (Santa Clara, CA, USA).

After transfection, cells were incubated for 5 d. The Idh2 knockdown cells were re-seeded at 30,000 cells/well onto an XF24 microplate 24 h prior to experimentation. The day before the oxygen consumption rate experiment, the sensor cartridge was calibrated using the calibration buffer (Seahorse, Agilent, Santa Clara, CA) overnight at 37 °C. One h before OCR measurement, transfected cells were washed twice with XF media and incubated in a non-CO₂ incubator at 37 °C. Oxygen consumption rate (OCR) measurements were performed using the Mito Stress Test (Seahorse, Agilent, Santa Clara, CA), according to the manufacturer’s protocol using the Seahorse XF96 Analyzer (Agilent, Santa Clara, CA) as described previously^{42 (link)}. Oligomycin, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), antimycin A, and rotenone were reagents used to determine OCRs. All reagents were purchased from Sigma-Aldrich (St. Louis, MO).

The mitochondrial oxygen consumption rate (OCR) was measured using a Seahorse XF-24 analyzer (Seahorse Bioscience Inc., North Billerica, MA, USA) in 24-well plates. Hepatocytes were seeded at 2 × 10⁴ cells per well 24 h before the analysis. On the day before the OCR measurement, the sensor cartridge was placed into calibration buffer (Seahorse Bioscience) and incubated in a non-CO₂ incubator at 37 °C. Hepatocytes were washed and incubated in DMEM without sodium bicarbonate. The medium and mitochondrial OXPHOS inhibitors were adjusted to pH 7.4 on the day of the OCR assay. The basal OCR was measured three times, and three readings were taken after the addition of each mitochondrial OXPHOS inhibitor [oligomycin (2 µg/mL) and rotenone (1 µM)]. The basal and post-oligomycin OCRs were calculated by averaging the last three measurements after maintaining a steady state. Coupled respiration was expressed as the percent decrease from basal respiration. Additionally, carbonyl cyanide m-chlorophenyl hydrazone (CCCP; 5 µM) was used to measure maximal mitochondrial respiration of the cells. OCR was automatically calculated and recorded by the sensor cartridge and the Seahorse XF-24 software.