Xf24 extracellular flux analyser

OCRs and ECARs were measured using an XF24 Extracellular Flux Analyser (Seahorse Bioscience, North Billerica, MA, USA). HeLa cells (15×10³) transfected with siCtrl or siOPA1 were plated on XF24 microplates 3 days before OCR measurements. Dual-analyte sensor cartridges were soaked in XF Calibrant Solution (Seahorse Biosciences) in 24-well cell-culture microplates overnight at 37°C to hydrate. Approximately 1 h prior to experimentation, injection ports on the sensor cartridge were filled with oligomycin (1 µM), FCCP (1 µM), and rotenone (1 µM) plus antimycin A (1 µM). Plates were then loaded into the XF24 instrument for calibration. For measuring oxygen consumption, the DMEM of HeLa cells was replaced by DMEM supplemented with NaCl (143 mM), Phenol Red (3 mg/ml), glucose (10 mM), glutamine (2 mM) and pyruvate (2 mM) at pH 7.4, and the plates were maintained at 37°C 1 h prior to experimentation. Plates were then loaded into the Seahorse XF24 analyser following the manufacturer's instructions.
ATP measurements in HeLa cells were determined using an ATP Colorimetric/Fluorometric Assay kit (Abcam). Intracellular ATP levels were determined using the colorimetric assay, following the manufacturer's instructions, on 1.10⁶ HeLa cells transfected with control siRNA or OPA1 siRNA. ATP content was measured in duplicate (570 nm) and calculated per microgram of protein.

Oxygen-consumption rate of live cells in a 24-well plate was measured in real time using a Seahorse Bioscience XF24 extracellular flux analyser. Cell number optimization was determined on 28,000 cells/mL which were seeded and grown for 24 h to 70–100% confluence before the metabolic flux analysis. Then, cell growth medium was replaced with XF assay medium (pH 7.4, Seahorse Biosciences) supplemented with 2 mM glutamine and 1 mM sodium pyruvate. Prior to the cell-respiration measurement, cells were incubated for 1 h at 37 °C without CO₂. Basal oxygen consumption, maximal respiratory capacity and non-mitochondrial oxygen consumption were determined using the XF Cell Mito Stress Test Kit (Agilent Technologies, UK). The inhibitors of mitochondrial respiration, including oligomycin, carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) and rotenone/antimycin were auto-injected into the experimental wells after basal measurements. Oligomycin used to inhibit ATP synthase, FCCP used as a protonophore, Rotenone and antimycin blocked mitochondrial respiration of electron transport chain. Cell number determined by haemocytometer was used to normalize the oxygen-consumption rate values between wells (n = 4 for each treatment) and treatments.