2.3. Cell Viability Assay

The 5-azacytidine (Merck KGaA, Darmstadt, Germany) concentration that reduced the viability of OSCCs and CSCs by 50% (IC₅₀) was determined by means of the colorimetric MTT (3-(4,5-dimethylthiazol 2yl)diphenyltetrazolium bromide) assay [29]. Cellular viability was evaluated by the ability of mitochondria to reduce the yellow MTT reagent to a purple formazan product. OSCCs and CSCs were seeded in 96-well plates (10 × 10³ cells per well) in DMEM F-12, respectively, with 10% FBS and 2% FBS, 20 ng/mL hrEGF and 10 ng/mL bFGF; cells were incubated at 37 °C in a humidified atmosphere with 5% CO₂. After 24 h, increasing concentrations of 5-azacytidine (0.1, 0.2, 0.4, 0.8, 1.0 μM for OSCCs and 0.1, 0.5, 0.4, 1.0, 1.5, 2.0 μM for CSCs) were added and cells were incubated for further 24 h at 37 °C and in a humidified atmosphere with 5% CO₂ [26]. The experiments were repeated in triplicate. Both OSCCs and CSCs were compared with the respective control. Cells were then incubated with 5 mg/mL of MTT for 3 h at 37 °C; at the end of the treatment, the medium containing MTT was removed and 100 μL of dimethylsulphoxide (Merck KGaA, Darmstadt, Germany) were added to each well. The number of viable cells was correlated to the intensity of the purple formazan product, measured at 570 nm, by means of a microplate reader (Synergy HT, Biotek, Winooski, VT, USA). The ratios between the absorbance values (Abs) measured for drug-treated cells (named OSCC-5Aza and CSC-5Aza) and those of untreated ones (named OSCC-Ctrl and CSC-Ctrl) were calculated and reported in viability curves, as percentages of viable cells against the inhibitory effect of the treatments on the cellular mitochondrial activity: (Abs_OSCC-5Aza/Abs_OSCC-Ctrl) × 100 and (Abs_CSC-5Aza/Abs_CSC-Ctrl) × 100. The maximum of cellular metabolic activity (100%) was assumed for untreated control samples. The drug concentration that reduced the viability of cells by 50% (IC₅₀) was determined by applying a linear regression between triplicate cellular data points and the 5-azacytidine concentration range (OriginPro 2018b software, OriginLab Corporation, Northampton, MA, USA).