O2k oxygraph chamber

Cardiac mitochondria were isolated as previously described [20 (link)]. Mitochondria (200 μg) were introduced into O2K oxygraph chambers (Oroboros Instruments, Innsbruck, Austria) to evaluate their oxygen consumption in presence of different compounds. In a first series of experiments, pyruvate (5 mM), glutamate (5 mM) and malate (2 mM) were added. After signal stabilization, ADP (0.5 mM) was injected in order to measure oxygen consumption when oxidative phosphorylation occurs. The second series of experiments consisted in adding palmitoylcarnitine (20 μM) in presence of malate (2 mM). Then ADP (0.5 mM) was injected into the chambers in order to evaluate respiration with substrates subjected to β-oxidation.

NCMs were seeded at 4 × 10⁵ cells/well in 6 well-plates (1 plate per condition) in culture medium with serum. Then, cells were serum-deprived for 24 h and treated with PBS (control) or Iso at 10 µmol/L for 24 h and 48 h in serum-free medium. A pre-treatment of MitoQ (1 µmol/L, 2 h), or DMSO (as control) was also used. At the end of treatment, NCMs were gently rinsed twice with PBS. Pre-warmed trypsin solution was added into each well and incubated at 37 °C for 10 min. Once cells appeared detached, trypsin was inactivated by addition of medium containing serum and centrifuged at 800× g for 7 min. After removing the supernatant, the cell pellet was resuspended in pre-warmed medium with serum and count with Malassez cell.
NCMs (between 10⁶ and 1.5 × 10⁶ cells) were incubated into the O2K oxygraph chambers (Oroboros Instruments, Innsbruck, Austria) at 37 °C under constant stirring. After a 15 min stabilization leading to resting respiration, oligomycin A (5 nmol/L) was added to measure leak respiration for 5 min. Then, carbonyl cyanide m-chlorophenyl hydrazine (CCCP) pulses (0.5 to 2.5µmol/L steps) were performed until maximal oxygen consumption was achieved. Non-mitochondrial oxygen consumption was obtained after antimycin A (AA) (2.5 µmol/L) injection for 5 min.

Mitochondria respiratory rates were measured into the O2K oxygraph chambers (Oroboros Instruments, Innsbruck, Austria) at 37°C in the respiration medium MiR06 (0.5 mM EGTA, 3 mM MgCl₂, 60 mM K-lactobionate, 20 mM taurine, 10 mM KH₂PO₄ 20 mM Hepes, 110 mM sucrose and 1 g/l bovine serum albumin fatty acid-free, 280 U/ml catalase (pH 7.1)).
Drp1 clones were grown in standard conditions and 1 × 10⁶ viable cells were transferred into oxygraph chambers. Oxygen consumption reached a steady state level indicating Basal respiration. The addition of oligomycin (0.5 μM) resulted in Leak respiration. Subsequently, the mitochondrial proton gradient was lost by stepwise titration (0.5 μM each step) of the uncoupler FCCP (Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone) until the maximum respiration was achieved. The addition of 0.5 μM rotenone and 2.5 μM antimycin A (AA) blocked mitochondrial respiration, showing residual oxygen consumption. The initial addition of pyruvate (10 mM) and malate (2 mM) was performed to test the integrity of the plasma membrane. Oxygen fluxes were corrected by subtracting residual oxygen consumption from each steady state.

Mitochondria respiratory rates were measured into the O2K oxygraph chambers (Oroboros Instruments, Innsbruck, Austria) at 37 °C in the respiration medium MiR06 (0.5 mM EGTA, 3 mM MgCl2, 60 mM K-lactobionate, 20 mM taurine, 10 mM KH2PO4 20 mM Hepes, 110 mM sucrose and 1 g/L bovine serum albumin fatty acid-free, 280 U/mL catalase (pH 7.1)) [37 (link),41 (link),42 (link),47 (link)]. Basal respiration was observed when the signal of oxygen consumption was stable, while the leak respiration was induced after the addition of oligomycin (0.5 µM). Stepwise titration (0.5 μM each step) of the uncoupler carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) induced the progressive release of the proton gradient until maximal respiration was achieved. The residual oxygen consumption was evaluated by blocking mitochondrial respiration with the addition of 0.5 μM rotenone and 2.5 μM antimycin A (AA), and this value was subtracted by each steady-state. Coupling efficiency was the ratio: (Basal Respiration - Leak respiration)/Basal respiration.