For cells in monolayers, the Seahorse Bioscience XFe 96 Extracellular Flux Analyser (Seahorse Bioscience, North Billerica, USA) was the instrument of choice, and for cells in suspension such as blood cells the Oroboros O2k (Oroboros Instruments, Innsbruck, Austria) was used. Respiratory measurements using Oroboros O2k were performed in stirred (750 r.p.m.) 2 ml glass chambers at 37 °C. The media MiR05 (sucrose 110 mM, HEPES 20 mM, taurine 20 mM, K-lactobionate 60 mM, MgCl2 3 mM, KH2PO4 10 mM, EGTA 0.5 mM and bovine serum albumin 1 g l−1, pH 7.1) was used in all experiments16 (link)17 . Data were recorded using the DatLab software version 4, 5 or 6 (Oroboros Instruments). Correction for instrumental background and air calibration was performed according to the manufacturer's instructions.
All experiments with platelets were performed with cell concentrations of 200 × 106 cells per ml and all experiments with PBMCs with 5 × 106 cells per ml. In experiments with human heart fibres, ∼10 mg of tissue was used in each run. To inhibit mitochondrial CI, rotenone (2 μM) was used and to inhibit mitochondrial complex III, antimycin A (1 μg ml−1) was used. ATP synthase was inhibited using oligomycin (1 μg ml−1), evaluating the contribution of respiration independent of ADP phosphorylation. Maximum uncoupled respiration of the electron transport system was induced by titration of the protonophore carbonyl cyanide FCCP until no further increase in respiration was detected. The test compound or control substances (succinate, dimethyl succinate, monomethyl succinate, malonate, dimethyl malonate or dimethylsulphoxide (DMSO)) were dosed as indicated in each figure.
Respirometric measurements in fibroblasts were performed using a Seahorse Bioscience XFe 96 Extracellular Flux Analyzer. The day before the experiment, fibroblasts were seeded out at 25,000 cells per well in cell growth medium in collagen-coated 96-well plates and kept at 37 °C and 5% CO2 overnight. Before the experiment, the growth medium was replaced by XF-Base Medium containing 2 mML -glutamine, 5 mM sodium pyruvate and 10 mM glucose (pH 7.4) and the cells were kept at 37 °C 1 h at atmospheric O2 and CO2. Oxygen consumption was measured at routine state and after addition of 500 μM of NV241 or NV189, its vehicle DMSO, dimethyl succinate or disodium succinate, followed by different concentrations of FCCP (0.125, 0.5, 1.0 and 1.5 μM), 2 μM rotenone and 1 μg ml−1 antimycin A. After FCCP and drug addition, the first data point was generally used, if not another data point was clearly higher, and for the remaining states the last data point before the subsequent addition was used. The FCCP dosing resulting in the highest uncoupled respiration was chosen for analysis for each experiment with each cell line and treatment.
All respirometric measurements, with the exception of the human heart fibre data, were corrected for non-mitochondrial oxygen consumption, obtained after the addition of antimycin A.
All experiments with platelets were performed with cell concentrations of 200 × 106 cells per ml and all experiments with PBMCs with 5 × 106 cells per ml. In experiments with human heart fibres, ∼10 mg of tissue was used in each run. To inhibit mitochondrial CI, rotenone (2 μM) was used and to inhibit mitochondrial complex III, antimycin A (1 μg ml−1) was used. ATP synthase was inhibited using oligomycin (1 μg ml−1), evaluating the contribution of respiration independent of ADP phosphorylation. Maximum uncoupled respiration of the electron transport system was induced by titration of the protonophore carbonyl cyanide FCCP until no further increase in respiration was detected. The test compound or control substances (succinate, dimethyl succinate, monomethyl succinate, malonate, dimethyl malonate or dimethylsulphoxide (DMSO)) were dosed as indicated in each figure.
Respirometric measurements in fibroblasts were performed using a Seahorse Bioscience XFe 96 Extracellular Flux Analyzer. The day before the experiment, fibroblasts were seeded out at 25,000 cells per well in cell growth medium in collagen-coated 96-well plates and kept at 37 °C and 5% CO2 overnight. Before the experiment, the growth medium was replaced by XF-Base Medium containing 2 mM
All respirometric measurements, with the exception of the human heart fibre data, were corrected for non-mitochondrial oxygen consumption, obtained after the addition of antimycin A.
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