2.4. Intracellular real-time GSH determination with RT-probe

Lyophilized RealThiol (RT) probe was kindly provided by Jin Wang and Xiqian Jiang (Jiang et al., 2017). 5 mM stock solution was prepared by dissolving lyophilized RT-probe in DMSO and stored aliquoted at −80 °C. Immediately before starting the assay, working concentration of 1 μ M was adjusted by dilution with DMSO (50 times) and fresh medium (100 times). 1 mL working concentration of RT-probe was sufficient for 2 × 10⁵ cells.

HaCaT and HaCaT/SM cells were diluted to yield 1 × 10⁵ cells per mL and one mL each was transferred to a new reaction tube. RT-probe was diluted to working solutions directly to the cell suspension. The suspensions were mixed and stained for 10 to 15 min at room temperature. Afterwards, 50 μL of stained cell suspension was transferred into a black 96-well plate with a clear bottom (Greiner). Baseline was recorded every 2 min for 12 cycles using a plate reader infinite M200 Pro at 37 °C and 5 % CO₂ (Tecan). Cells were exposed to 100 μM SM and recording was continued until 400 min. Parameters determined were F₄₀₅ (405 nm excitation, 430­470 nm emission) and F₄₈₈ channel (488 nm excitation, 510–660 nm emission) and GSH concentration was determined as F₄₀₅/F₄₈₈ ratio.