Dcf da probe

For the measurements of intracellular ROS production, HDFs were seeded onto 96 well plates at a density of 2 × 10³ per well and analyzed 24 hours post-treatment with the compounds. The medium was removed and replaced with PBS, and the cells were incubated for 30 minutes with a 10 mM 2ʹ7’-dichlorofluorescein diacetate (DCF-DA) probe (Abcam). Conversion into fluorescent DCF was induced by further incubation of the cells in DMEM/10% FBS in a CO₂ incubator for 15 minutes. The cells were fixed with 4% formaldehyde/PBS, washed with PBS, and relative fluorescence intensity was recorded in a microplate reader (SpectraMax iD5 Microplate Reader, Molecular Devices) at Ex/Em 485/535 nm.

For the measurements of intracellular ROS production, HDFs were seeded onto 96 well plates at the density of 2×10³ per well and analyzed 72 hours post-treatment. The media was removed, replaced with PBS and the cells incubated for 30 minutes with a 10 μM 2’7’-dichlorofluorescein diacetate (DCF-DA) probe (Abcam). Conversion into fluorescent DCF was induced by further incubation of the cells in DMEM/10% FBS in a CO₂ incubator for 15 minutes. The cells were fixed with 4% formaldehyde/PBS, washed with PBS and relative fluorescence intensity was recorded in a microplate reader (SpectraMax iD5 Microplate Reader, Molecular Devices) at Ex/Em 485/535 nm and adjusted to an equal number of cells. For statistical analysis, the data comparing variation between skin types for each applied factor were validated using one-way Analysis Of Variance (ANOVA) with post-hoc Tukey HSD test, n = 3, experimental replicates. The graphs represent Mean ± SEM, with statistically significant outputs *p < 0.05, **p < 0.01, ***p < 0.001.