Measurements of cellular oxygen consumption rate

Cellular oxygen consumption rate analyses were performed using the Agilent Technologies Seahorse XFp analyzer (8 chambers; Seahorse Bioscience, North Billerica, MA, USA) and the Agilent Seahorse XFp Cell Mito Stress Test Kit. On day 1 of the assay, myoblasts were seeded in GM (4 x 10³ cells/chamber) in six chambers of the Seahorse XFp Cell Culture Miniplate. The two remaining chambers were kept as negative controls (no cells). Cells were grown in GM at 37°C and 6% CO₂ in a humidified incubator. The culture medium was removed on day 2 and replaced with fresh GM (with or without carnosine). After completion of carnosine and H₂O₂ treatment periods (at 90% confluence), the GM was removed (leaving 20 μL of GM to avoid cell dryness) and replaced with 200 μL of assay medium (HEPES base medium (5 mM) containing 1 mM sodium pyruvate, 2 mM glutamine and 25 mM glucose; pH 7.4). The cell culture miniplates were incubated 1 h, at 37°C and without CO₂. Components of the Cell Mito Stress Test Kit were then used to assess oxygen consumption rate. Basal respiration was first measured, followed by sequential injection of modulators of components of the mitochondrial electron transport chain (oligomycin (1 μM), carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP; 2 μM) and a mixture of rotenone and antimycin A (0.5 μM)). At the end of each assay, cells were collected to determine total cellular proteins. Data were analysed using the Seahorse Wave Desktop Software and are expressed as oxygen consumption rate (OCR; pmol/min/μg protein). The mean values of 5 independent experiments were used for statistical analyses. These analyses allowed for the determination of the effect of treatments on basal respiration, ATP-linked respiration, maximal respiration, spare respiratory capacity, proton (H+) leak and non-mitochondrial respiration.