2.6. Inhibition of Cell Viability Assay

The cytotoxicity of the compounds was determined by colourimetric microculture assay (MTT assay). The cells were harvested from culture flasks by trypsinisation and seeded into Cellstar 96-well microculture plates (Greiner Bio-One, Practical Mediscience Pte Ltd, Singapore, Singapore) at the seeding density of 6 × 10⁴ cells per mL. After the cells were allowed to resume exponential growth for 24 h, they were exposed to drugs at different concentrations in media for 72 h. The drugs were diluted in complete medium at the desired concentration, and 100 μL of the drug solution was added to each well and serially diluted to other wells. After exposure for 72 h, drug solutions were replaced with 100 μL of MTT in media (5 mg mL⁻¹) and incubated for an additional 45 min. Subsequently, the medium was aspirated, and the purple formazan crystals formed in viable cells were dissolved in 100 μL of DMSO per well. Optical densities were measured at 570 nm with a microplate reader. The number of viable cells was expressed in terms of treated/control (T/C) values by comparison to untreated control cells, and 50% inhibitory concentrations (IC₅₀) were calculated from concentration-effect curves by interpolation. The evaluation was based on means from at least three independent experiments, each comprising six replicates per concentration level.