Amplex ultrared

Diaphragm mitochondrial hydrogen peroxide production from permeabilized muscle fibers was measured continuously using a Fluorolog-3 spectrofluorometer (HORIBA, Kyoto, Japan). Measurements were performed at 37 °C using the Amplex UltraRed (Molecular Probes, Eugene, OR, USA) (10 µM)/horseradish peroxidase (HRP) (1 U/mL) system as described [45 (link)].

The Chinese hamster ovary cell line 51D1.3 (herein called CHO) and a stable transfectant expressing a soluble form of human P450 oxidoreductase (51D1.3/sPOR, herein called CHO/POR) (Gu et al., 2009 (link)), were grown in spinner flasks in αMEM plus 5% fetal bovine serum. Late log phase cultures were harvested by centrifugation, resuspended at 5 × 10⁵ cells/mL in phenol red-free high-glucose DMEM (Invitrogen) supplemented with 4 mM Glutamax-1, 1 mM sodium pyruvate and 5% FBS. Stirred cell suspensions (2 mL) were transferred to an OXYBOROS O2K oxygraph for simultaneous polarographic measurement of non-respiratory O₂ consumption and fluorimetric measurement of H₂O₂ generation as previously (Hunter et al., 2012 (link)). Bovine liver superoxide dismutase (10 U), horseradish peroxidase (10 U), Amplex UltraRed (50 nmol) and rotenone (1 nmol), all from Molecular Probes Invitrogen (Eugene, OR), were injected and cumulative drug additions (from saline stock solutions) were made every ∼5 min once endogenous rates had stabilized. Signals were recorded every 2 s, and flux derivatives were estimated ∼2 min after each addition using a Savitzky–Golay smoothing filter (40 data point window) in DATLAB version 4.3.

OCR and the rate of mitochondrial hydrogen peroxide production were simultaneously determined using the O2k (OROBOROS Instruments, Innsbruck, Austria) and O2k-Fluo LED2-Module Fluorescence-Sensor Green. Following rapid dissection, the dorsal hippocampus (2–3 mg) was suspended in ice cold Buffer X (7.23 mm K₂EGTA, 2.77 mm CaK₂EGTA, 20 mm imidazole, 0.5 mm DTT, 20 mm taurine, 5.7 mm ATP, 14.3 mm PCr, 6.56 mm MgCl₂–6H2O, and 50 mm K-MES with a pH of 7.1) and permeabilized with saponin (50 µg/ml) for 30 min followed by 3 × 5 min washes in ice-cold wash buffer Z [105 mm K-MES, 30 mm KCl, 10 mm K₂HPO₄, 5 mm MgCl₂–6H2O, 0.5 mg/ml bovine serum albumin, 0.1 mm EGTA (pH 7.1)]. O2k measurements were obtained in buffer Z media at 37 °C containing 10 µm Amplex UltraRed (Molecular Probes, Eugene, OR), 1 U/ml horseradish peroxidase, and 5 U/ml SOD. Rates of respiration were determined using sequential additions of substrates and inhibitors as follows: glutamate (10 mm), malate (2 mm), pyruvate (5 mm), ADP (5 mm), succinate (10 mm), rotenone (1 µm), antimycin A (1 µm), and TMPD (0.5 mm) immediately followed by ascorbate (5 mm). Antimycin A values were used to normalize respiration measurements to account for nonmitochondrial oxygen consumption. Data for OCR were normalized by milligrams of hippocampal wet weights.