2.3. Cytotoxicity Assay

MTT assay was performed as described in the previous study [52]. The copper complex was prepared as stock solutions, to suit the universal 96-well plate map, at different concentrations in nM or µM, dissolved in 100% DMSO (Sigma-Aldrich, St. Louis, MO, USA) or dH₂O. Working solutions were prepared in the culture medium, at a final DMSO concentration of 0.02%, where DMSO was used as the solvent, and added to the wells 24 h after seeding of 5 to 7 × 10⁴ cells per well in 200 μL of fresh culture medium. DMSO (0.02%) was used as the solvent control. Light microscopic cytological changes were monitored and photographed following exposure to different concentrations of the complex for 24 and 48 h using an inverted microscope (Carl Zeiss, Jena, Germany). After the respective periods of treatment, 20 μL of MTT solution [5 mg/mL in phosphate-buffered saline (PBS)] was added to each well, and the plates were wrapped with aluminum foil and incubated for overnight at 37 °C. The purple formazan crystal that formed was dissolved in 100 μL of 100% DMSO. The absorbance of the color was monitored at 570 nm (measurement) and 630 nm (reference) using a 96-well microplate reader (Bio-Rad, Hercules, CA, USA). Data were collected for four replicates each and used to calculate the median effect dose, i.e., IC₅₀ (Dm value), sigmoidity of the dose-effect curve (m value) and linear correlation coefficient of the median-effect plot (r value), using Calcusyn software (Biosoft, Cambridge, UK).