Cytotoxicity Evaluation of Anti-Cancer Agents

A375, SK-MEL28, FM55P, and FM55M2 melanoma cells and normal human keratinocytes (HaCaT) cells were placed on 96-well plates (Nunc, Roskilde, Denmark) at a density of 2–3 × 10⁴ cells/mL. The next day, the culture medium was removed and cells were exposed to serial dilutions of paclitaxel, docetaxel, and betulinic acid in a fresh culture medium. Cell viability was assessed after 72 h by means of the MTT method. The 72 h incubation time is the average doubling time for all melanoma cell lines tested. In the case of the A375 line, this time is the shortest 6–12 h [55 (link)], for the SK-Mel28 it is 17.5 h [56 (link)], but in the case of the FM55M2 and FM55P lines, most of the experiments encountered are the incubation time of 72 h [37 (link),38 (link),57 (link)]. After 72 h incubation, cells were incubated for 3 h with MTT solution (5 mg/mL, Sigma-Aldrich, USA). Formazan crystals were solubilized overnight in sodium dodecyl sulfate (SDS) buffer (10% SDS in 0.01 N HCl) and the product was determined spectrophotometrically by measuring absorbance at 570 nm wavelength using a microplate spectrophotometer (Ledetect 96, Labexim, Lengau, Austria). Each experiment was repeated three times.