Simultaneous Determination of NAD+/NADH and ΔΨ

NAD⁺/NADH and ΔΨ were determined simultaneously using a QuantaMaster Spectrofluorometer (QM-400, Horiba Scientific, Kyoto, Japan) as previously described (25 (link)), with some modifications. Experiments were performed with tissue mitochondria (20µg) in a 200µL reaction volume at 37°C. Assay buffer was Buffer D supplemented with Cr (5mM) and tetramethyl rhodamine methyl ester (TMRM; 0.2 µM). Mitochondria were stimulated using the creatine kinase clamp and ΔG_ATP was titrated via PCr additions (6, 15, 21mM). Oligomycin (0.02µM) was added to inhibit ATP synthesis, and cyanide (CN, 10mM) was added to induce 100% reduction of the matrix NADH pool, followed by isocitrate (5mM) to induce 100% reduction of the matrix NADPH pool. NAD⁺/NADH was detected at Ex/Em: 350/450 and expressed as a percentage reduction of the CN value as previously described (25 (link)). TMRM-derived ΔΨ was quantified by taking the fluorescence ratio of Ex/Em 576/590 to 551/590 and converting this to mV values using a tissue-specific standard curve determined as previously described (23 (link)).