2.3. Cell Cytotoxicity Assay and Half-Maximal Cytotoxic Concentration (CC50) Determination

Cell viability was assessed using CellTiter 96 AQ_ueous One Solution reagent and cell proliferation assay (Promega, Madison). This method determined the number of viable cells based on conversion of formazan product from 3-(4,5-dimethylthazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolim by nicotinamide adenine dinucleotide phosphate (NADPH) or reduced NADPH (NADH) generated in living cells. A549 cells were plated on 96-well clear-bottom plates (2.0 × 10⁴ cells/well). Serial dilutions (three-fold) of each compound were added to cells and, 48 h after drug treatment, the viability reagent was added and incubated for 15 min (37 °C and 5% carbon dioxide [CO₂]). Absorbance was measured at 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader (SpectraMax ABS Plus, Molecular Devices, Sunnyvale, CA, USA). The resulting optical densities were normalized with dimethylsulfoxide (DMSO); the vehicle control group was adjusted to 100%. CC₅₀ was determined using Prism (GraphPad, San Diego, CA, USA).