2.2.1. Mito Stress Test

To evaluate mitochondrial function, the Mito Stress test was performed using a Seahorse XF96 Cell Analyzer according to the manufacturer's recommendations. Briefly, myoblasts from patients and controls were seeded to Seahorse XF96 cell culture microplates (2.5 × 10<sup>4</sup> cells per well) in skeletal muscle cell growth medium supplemented with 10% fetal bovine serum (FBS). After a 24-h incubation at 37 ◦C, the cells were washed twice with a pre-warmed assay medium (XF base medium supplemented with 10 mM glucose, 2 mM glutamine, and 1 mM sodium pyruvate; pH 7.4).

Oxygen-consumption rate (OCR) values were simultaneously measured following sequential injections of inhibitors of mitochondrial oxidative phosphorylation: (I) oligomycin (2 µM), an ATP synthase inhibitor; (II) carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP, 2 µM), an uncoupler; (III) antimycin A (0.5 µM) (inhibitor of complex III) and (IV) rotenone (0.5 µM) (inhibitor of complex I), inhibiting uncoupled respiration. Key parameters of mitochondrial function such as basal respiration (BR), ATP production rate (ATP-R) and spare respiratory capacity (SRC) were analyzed using the above-described measurements. The data was normalized to cell numbers by measurement of Hoechst dye staining of nuclei with excitation and emission wavelengths 355 nm and 465 nm using a Tecan Infinite TM M1000 and plotted as OCR (pmol/min/U fluorescence/well ± SD), accordingly.
