Intracellular ROS Assay in Keratinocytes and Fibroblasts

To assay the capacity of C. mas L. fruit extract to generate intracellular level of reactive oxygen species in keratinocytes (HaCaT) and fibroblasts (HDF) cells, the method of Grauzdytė et al. [81 (link)] with some modifications, was used. In this method, the fluorogenic dye 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA; (Sigma Aldrich, Sant Louis, MO, USA) was used. Cells were seeded in 96-well plates at a density of 1 × 10⁴ cells per well and initially cultured before the experiment for 24 h. After this time, the DMEM medium was removed and cells (HaCaT and HDF) were treated with C. mas L. extracts dissolved in DMEM at concentrations of 1, 5, 10% for another 24 h. Then, DMEM medium with extracts was changed on 10 µM H₂DCFDA in serum-free DMEM medium in each well. Then, 5 mM hydrogen peroxide (H₂O₂; in a final concentration of 500 µM) dissolved in DMEM without serum was immediately added to the tested samples and the positive sample. After 60 min of incubation, the measurement was taken at an excitation wavelength of λ = 485 nm and an emission wavelength of λ = 530 nm using a microplate reader (FilterMax F5, Thermo Fisher Scientific, Waltham, MA, USA). This analysis included three independent experiments, with each sample being tested in triplicate.