2.6. DCFH-DA-Assay for ROS Detection

B16F10 cells were seeded at a density of 3 × 10⁴ cells per well in 24-well plates in duplicates and then incubated overnight at 37 °C. After 24 h, cells were treated with 0–5 mM ascorbate for different time periods. Three h before the end of treatment, 50 µL of medium were removed from the positive control wells and replaced with 50 µL 10 mM tert-butyl hydroperoxide (TBH; Sigma-Aldrich) solution to achieve a final concentration of 1 mM. Two, three, and four h after the start of treatments, medium was removed, washed with 500 µL of PBS, and cells were harvested with 150 µL trypsin at 37 °C for 5 min. For each well, 1 mL of medium was added to stop the trypsin reaction, cells were centrifuged (149× g, 5 min), and the supernatant was discarded. A working solution with 5 µM in phenol red-free, FCS-free medium was prepared from the 40 mM 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Sigma-Aldrich) stock solution, and the cell pellets were resuspended in 1 mL of this solution. After intracellular esterases increase the polarity of the molecule by deacetylation and prevent it from leaving the cell, the green fluorescent dichlorofluorescein (DCF) is formed by oxidation by various ROS [39]. After incubation at 37 °C for 30 min in the dark, the cells were centrifuged, the supernatant was discarded, and the cells were covered once again with PBS. Subsequently, pellets were resuspended in 400 µL of phenol red-free, FCS-free medium and were transferred to a flow cytometry tube. Flow cytometric analysis followed with absorbance at 488 nm and detection of emission at 530 nm (NovoCyte^® 2060R flow cytometer, Agilent Technologies Inc.). For this purpose, 10,000 events per sample were measured and the experiment was performed three times, independently.