4.3. Mitochondrial Respiration

Measurements of cellular oxygen consumption were performed using an XFe96 Extracellular Flux Analyzer, using the Seahorse XF Mito Stress Test kit (Seahorse Bioscience, North Billerica, MA, USA). H9C2 cardiomyoblasts were incubated for 5–6 days in culture medium in the presence or absence of doxycycline, followed by overnight incubation at 25,000 cells/well in Seahorse 96-well culture microplates. One hour before measurement, medium was replaced by DMEM (Sigma, D5030, Zwijndrecht, Netherlands) containing 25 mM glucose (Sigma), 1 mM sodium pyruvate (Lonza, Basel, Switzerland), and 2 mM L-glutamine (Life Technologies, Bleiswijk, The Netherlands) and cells were incubated in a non-CO₂ 37 °C incubator. Mitochondrial respiration was measured before (routine respiration) and after 1.5 μM oligomycin (leak respiration), 1 μM carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP) (maximal uncoupled respiration), 2.5 μM antimycin A and 1.25 μM rotenone (residual respiration). Experiments were performed with 8–10 wells per condition, and subsequently averaged. Oxygen consumption rate (OCR) values were adjusted for residual respiration and cell count using the CyQUANT Cell Proliferation Assay Kit (Thermo Fischer Scientific, Bleiswijk, Netherlands) according to the supplier’s protocol. To compare between plates, control routine respiration was set to 100.