4.3. Cell Growth Inhibition Assay

To estimate cell viability, the MTT assay was used. HCT116 and H460 cells were seeded in 24-well plates with 2 × 10⁴ cells per well. After 24 h of incubation at 37 °C in 5% CO₂ atmosphere, unsymmetrical bisacridines or reference compounds (irinotecan/cisplatin) were added at concentrations up to 10 µM for UAs and up to 200 µM for reference compounds. After 72 h of incubation, 200 µL/well of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Abcam, Cambridge, Great Britain) at a concentration of 4 mg/mL was added and incubated for 3 h at 37 °C. Next, the culture medium from each well was removed, the formazan crystals were dissolved in DMSO, and absorbance at 540 nm was measured. The concentrations of drugs required for inhibition of cell growth by 90% (IC₉₀) for UAs and 50% (IC₅₀) for reference compounds compared with untreated control cells were calculated from the curves plotting survival as a function of dose. Results were obtained from at least four independent experiments (n = 4).