Mitochondrial Respiratory Function Analysis

The oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) were measured by mitochondria stress test using an XFe96 extracellular flux analyzer (Seahorse Bioscience, North Billerica, MA, USA), as previously described [25 (link)]. Briefly, 5.0 × 10⁴ cells were seeded in XFe96-well plates overnight in respective complete media, and the OCR and ECAR were measured in Seahorse XF DMEM pH 7.4 (Agilent Technologies) that was supplemented with 10 mM D-glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate. As indicated, the cells were treated with the following pharmacological modulators of mitochondrial respiratory chain proteins: 1.5 µM oligomycin, 2.0 µM carbonyl cyanide p-tri-fluoromethoxyphenylhydrazone (FCCP), 0.5 µM antimycin A, and 0.5 µM rotenone (Seahorse bioscience). A total of three basal rate measurements were taken (0–14 min) prior to injection of the compounds, followed by three measurements of OCR/ECAR following the injection of each drug. OCR and ECAR readings were normalized to total protein levels (BCA protein assay, Pierce) in each well. Each cell line was seeded in 96 wells per experiment and replicate experiments were carried out at least three times.