2.7. Cell Viability: MTS Cytotoxicity Assay

Human melanoma cells MUG Mel-1, MUG Mel-2 cells and murine melanoma B16V were seeded at a density of 1 × 10⁴ cells/well in a 96-well plate and maintained under standard growth condition. After overnight incubation, cells were treated as follows: (i) AdV-D24 (0.1, 1, 10, 100 VP/cell), (ii) AdV-D24-ICOSL-CD40L (0.1, 1, 10, 100 VP/cell), (iii) anti PD-1 (100 µg/mL), (iv) AdV5-D24 (100 VP/cell) combined with anti PD-1 (100 µg/mL), (v) Ad5V-D24-ICOSL-CD40L (100 VP/cell) combined with anti PD-1 (100 µg/mL). Cell viability was determined 96 h after treatment, by using CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) according to the manufacturer’s instructions (Promega, Madison, WI, USA). The absorbance was measured with a 96-well plate spectrophotometer (Victor Nivo^TM, PerkinElmer, Milano, Italy) at 490 nm. The experiments were independently performed three times and each treatment was performed in triplicate.