4.4. Cytotoxic Assay (MTT) for Adherent Cancer Cell Lines

The MTT colorimetric assay was conducted based on the original procedure proposed by Mosmann [27], with a few modifications. The cell lines (HeLa, PC-3, or LNCaP) were seeded at a concentration of 1.5 × 10⁴ cells/well (96-well flat-bottom culture plate). After 24 h incubation to allow cell adherence [28], supernatants were removed and compounds added at the testing concentrations. An untreated control and doxorubicin-treated cells (positive control) [29] were included. Cells were incubated for a further 48 h [28]. Later, supernatants were removed and cells were washed. An MTT solution in culture media was added to the cells (0.2 mg/mL) followed by 4 h incubation [2]. After this time, supernatants were rejected and DMSO was used to solubilize the MTT formazan product [28]. After 10 min incubation with shaking, absorbance was measured using STAT FAX 3200 at 545/630 nm. Cytotoxicity was calculated as the percentage of cellular metabolic viability inhibition using the following formula: