4.3. Cell Viability Assay and Combination Index Determination

Cell viability was assayed by the CyQuant reagent (ThermoFisher Scientific/Life Technologies) to quantify the number of living cells, using a nuclear dye that selectively binds to nucleic acids, emitting fluorescence. Cells were cultured at a density of 0.10/0.20 × 10⁵ per mL in 96 multi-well plates and incubated (24–48 h) in a medium containing the specified treatments. The cell viability assay was performed as described [46]. Briefly, the CyQuant mixture, containing the nuclear dye (CyQuant nuclear stain) and the suppressor of basal fluorescence (background suppressor), was added to the culture medium and incubated for 1 h at 37 °C. Fluorescence was measured at the excitation wavelength of 485 and 530 nm emission. Moreover, the results were expressed as the percentage of fluorescence of the untreated control using a microplate reader (Synergy HT BioTek, Milan, Italy). The experiments were performed in quadruplicate and repeated 3 times. Cells were photographed using the FITC filter (magnification 400×) by an inverted microscope (Axiovert 200 Zeiss, Jena, Germany).

The combination index (CI) values were calculated according to the Chou and Talalay mathematical model for drug interactions, as previously reported [4]. Dose-response curves, dose-effect analysis, and CI for the combination treatment groups were generated with the equations reported by Chou and Talalay using the CompuSyn software (freely available at: www.combosyn.com Accessed on 27 September 2021).