To obtain fluorescence spectra and to generate DHE fluorescent products, 20 μM DHE from DMSO stock solution was freshly prepared in 50mM PBS (0.9% NaCl, 50 mM Na2HPO4, pH 7.4). Final DMSO content was 0.1%. The stock of 2OH-E as previously described 5 (link) was generated by complete DHE oxidation for 6 hours in superoxide generating cell-free enzymatic system containing 10 mU/ml xanthine oxidase (Roche Molecular Biochemicals Indianapolis, Ind., USA) and 0.5 mM xanthine. Xanthine (50 mM; Sigma Chemical Co. St. Louis, Mo., USA) was prepared in 0. 9% NaCl as a stock solution and the appropriate volume was added to the reaction mixture to reach required concentration. Progression of DHE oxidation was analyzed by HPLC.
Fluorescence intensities were acquired using BioTek H1 96-well plate reader. For experiments with cultured cells black glass-bottom plates were used (BD Bioscience, USA) for all other experiments polypropylene black plates (Nunc Thermo, Denmark). The instrument was kept at 37 °C during the measurements.
Fluorescence intensities were acquired using BioTek H1 96-well plate reader. For experiments with cultured cells black glass-bottom plates were used (BD Bioscience, USA) for all other experiments polypropylene black plates (Nunc Thermo, Denmark). The instrument was kept at 37 °C during the measurements.
