Oxytherm

2 × 10⁶ H9c2 cells were seeded in 100 mm culture plate. After being grown to stable attachment, cells were preincubated with ginsenoside Rb₂, ginsenoside F1, ginsenoside Rc, and schisandrin A at final concentrations (20 μM) for 18 h before being exposed to t-BHP (100 μM) for 1 h. Cells were washed with PBS twice, subsequently collected by trypsinization followed by centrifugation, and resuspended in fresh medium. Respiratory activity was measured with a Clark-type oxygen electrode (Oxytherm, Hansatech Instruments, UK). An aliquot (1 mL) of suspended cells (2 × 10⁶ cells/mL) was placed in the air-tight liquid-phase oxygen electrode chamber. The system was maintained at 37°C. After equilibration, the slope of oxygen consumption in H9c2 cells was measured. Every 1 × 10⁶ cells oxygen consumption was calculated as the basic respiration rate of each group. The experiment was also performed in the presence of SIRT1 inhibitor, EX527 at final concentration of 20 μM to investigate whether the effect of ginsenoside Rb₂ can be prevented by SIRT1 inhibitor.

Oxygen consumption rates were measured using an Oxytherm (Hansatech) oxygen electrode as previously described (Schulz et al. 2007 (link); Lee et al. 2009 (link); Rauthan et al. 2013 (link)). A Pierce BCA Protein Assay Kit (Thermo Scientific) was used to measure protein concentration.

The medium was collected to detect glucose uptake, lactate production and pH. The glucose uptake and lactate production were assessed with glucose uptake assay kit and lactate assay kit (Sigma, USA) following manufacturer’s instructions. The oxygen consumption rate was measured using a Hansatech Oxytherm (Hansatech, King’s Lynn, UK).

Cardiac mitochondria were isolated from WT and DKO mice as described previously^{43 (link)} through differential centrifugation. Mitochondrial respiratory function was measured using an Oxytherm (Hansatech) as previously described.^{44 (link), 45 (link)} Mitochondrial H₂O₂ emission was measured using Amplex Red (Invitrogen, Paisley, UK) in isolated mitochondria (500 μg/ml) suspended in Miro5 respiration buffer, according to the manufacturer's instructions. Complex I activity was stimulated by the addition of 5 mM Glutamate, 5 mM Malate, and 1 mM Pyruvate, and complex II activity by the addition of 10 mM Succinate and 0.5 mM rotenone. Susceptibility to MPTP opening was investigated in isolated cardiac mitochondria using a calcium-induced mitochondrial swelling assay, as previously described^{45 (link)} with optical density measured by spectrophotometry (FLUOstar Omega, BMG Labtech, Cary, NC, USA) for 20 min. Treatment with the known MPTP inhibitor, cyclosporin-A (1.0 μmol/l), was used as a positive control.