4.3. MTT Assay

Cell viability was assessed using the MTT assay. A375 and SB2 melanoma cells were seeded at a density of 10,000 cells per well in 24-well plates and allowed to adhere overnight. Cells were then treated for 72 h with NO donors, either DETA-NONOate or S-nitrosoglutathione (GSNO), at concentrations of 20, 50, or 100 µM. To maintain consistent exposure, the culture medium was replaced every 24 h with a fresh medium containing newly prepared NO donors. Following the 72-h treatment period, 20 μL of MTT reagent (5 mg/mL in PBS) was added to each well, and plates were incubated at 37 °C for 3 h. After incubation, the medium was carefully removed, and 200 μL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan crystals. The absorbance was measured at 570 nm and 630 nm using a BioTek Synergy H1 microplate reader (Santa Clara, CA, USA). Cell viability was calculated as a percentage relative to untreated control wells.