3.6. HaCaT Cells Culture and Viability

Human epidermal HaCaT keratinocytes were obtained from ATCC^® and maintained in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin (10,000 U mL⁻¹), and grown as monolayer at 37 °C in a 5% CO₂ atmosphere. After three days the culture was approximately 80% confluent, cells being harvested with trypsin-EDTA 0.25% solution (GIBCO, Invitrogen, Grand Island, NY, USA). Then the cells were subcultured in 96-well plates (1.5 × 10⁴ cells well⁻¹), grown until 80% confluence, and subsequently exposed to the extract for 24 h at 37 °C. Interference with the mitochondrial activity was studied 24 h later by the MTT reduction assay as described in Gomes et al. [50]. Viable cells were calculated as a percentage of the negative control cells set at 100%, results corresponding to the mean ± SEM of three independent experiments performed in triplicate.