Alamar blue cell viability assay

To study the effects of quercetin, MST-312 and luteolin independently on cell viability and logarithmic growth, PA-1, A2780 cells, OVCAR3, A2780cisR and HCT116 cells were seeded at a density of 8×104 cells/mL, 2×104 cells/mL, 9×104 cells/mL, 2×104 cells/mL and 1.5×104 cells/mL, respectively, in 96-well cell culture plates. Post 24 h, cells were incubated in the presence of increasing concentrations of quercetin and MST-312 for 72 h. Effect of luteolin on cell viability was evaluated in PA-1 cells for 72 h. Negative control (no compound) and vehicle control (DMSO) were maintained. After 72 h, cells were incubated in the presence of 20% solution (in DMEM) of 0.15 mg/mL alamar blue reagent in 1X Phosphate Buffered Saline (PBS) (pH 7.4) (Gibco, Cat. No.10010023) for 4 h at 37°C and absorbance was measured at 570 nm and 600 nm wavelengths in BioTek Epoch2 microplate reader (Agilent, USA) with Gen5 software (Version 3.03). OSE cells were seeded at a density of 4.5×10⁴ cells/mL and the assay was performed as above. Percentage reduction of alamar blue reagent was calculated using the following formula;

Where:

% reduction values were normalised to the control and then used to determine half-maximal inhibitory concentration (IC50) values from logarithmic growth curve using GraphPad Prism software (Version 8).