4.4. In Vitro Cytotoxicity Assays

Cell cytotoxicity assays were performed using MTT (3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide) assays [9] and lactate dehydrogenase (LDH) assays [41] to determine the effects of pure compounds against human cell lines. Briefly, for LDH assays, confluent HaCaT cell monolayers in a 96-well plate was challenged with 50 µg/mL of pure compounds and plates were incubated for 24 h at 37 °C in the presence of 5% CO₂ and humidified conditions. Next, the supernatant was mixed with an equal volume of LDH kit reagents and data analysis was performed spectrophotometrically at 490 nm. For MTT assays, pure compounds were incubated at various concentrations with overnight grown confluent monolayers of human cells and plates were incubated at 37 °C for overnight at 95% humidity and 5% CO₂. After this incubation, 10 µL of MTT solution was added to each well and plates was incubated for 3–4 h at 37 °C. The MTT solution was withdrawn carefully and 100 µL of DMSO was added to dissolve the crystals formed by viable cells. The plates were incubated for 15 min at 37 °C and the absorbance determined at 540 nm immediately.