4.5. Cytotoxicity In Vitro Assay

To evaluate the toxic effects of the 1,4-naphthoquinone derivatives on mammalian cells, Vero cells were seeded at a density of 1.5 × 10⁴ cells/well in 96-well white culture plates. Twenty-four hours later, the cell cultures were incubated for 72 h at 37 °C with 1,4-naphthoquinone derivatives (series 1(a–i) and 2(a–j)) and Bz (500–1.9 µM) diluted in RPMI medium supplemented with 10% FBS. After incubation, the cell viability, measured by ATP level, was assessed by adding 20 µL/well of the CellTiter Glo (Promega Corporation, Madison, WI, EUA) solution. The luminescent signal was read on the FlexStation 3 reader (Molecular Devices, Sunnyvale, CA, USA). The concentration of compound that reduces 50% of mammalian cell viability (CC₅₀) was determined by linear regression. All treatments and controls were performed at low concentration (≤1%) of dimethyl sulfoxide (DMSO). At least three independent assays were performed in duplicate.