D6883 50mg

Tissues to observe the ROS signals were submerged in 10 µM 2′,7′–dichlorofluorescin diacetate (DCFDA, SIGMA-Aldrich, D6883-50MG, Darmstadt, Germany) solution in 10 mM Tris-Cl buffer (pH 7.4) for 10 min. The samples were briefly washed and mounted with distilled water. An Olympus BX21 model was used to obtain the DCFDA signal. Seedling number to measure DCFDA signal intensity and concluding were indicated in each figure legends.

The cell monolayers and spheroids were washed with 1xPBS (10010-023, ThermoFisher Scientific) and permeabilized with 20 µM of 2′,7′-Dichlorofluorescin diacetate (DCFDA) (D6883-50MG, Sigma-Aldrich) for 30 min at 37 °C and 5% CO₂. After washing the cells with 1 × PBS twice, they were treated with 1 µM of quercetin, 50 µM of quercetin, 1 mM of hydrogen peroxide (BP2633500, ThermoFisher Scientific), 1 mM of hydrogen peroxide; 1 µM of quercetin, 1 mM of hydrogen peroxide, and 50 µM of quercetin; 5 mM of N-Acetyl-L-cysteine (A9165-5G, Sigma-Aldrich), 5 mM of N-Acetyl-L-cysteine, and 1 µM of quercetin; 5 mM of N-Acetyl-L-cysteine and 50 µM of quercetin; or they were left untreated for 60 min at 37 °C and 5% CO₂. The oxygen-reactive species were quantified by measuring the fluorescence at 485 nm (excitation) and 535 nm (emission) using the transmitted light detection cartridge (5022671, Molecular Devices) in the SpectraMax i3x platform and normalized to the cell count or area, which were determined using the Mini MaxTM 300 Imaging Cytometer (5024062, Molecular Devices). The treatments were performed in sextuplicate, and the fold changes of the different treatments, relative to the untreated control sample, were calculated. The evaluated data were graphically processed in Excel.