4.6. Anti-melanoma Activity Measurement by MTT Assay

The B16F10 melanoma cells were cultured in a growth medium containing antibiotics (1%) and FBS (10%) at 37 °C. For cell viability measurement by MTT assay [58,59,60,61,62,63], B16F10 cells were seeded in 96-well plates at a density of 1 × 10⁴ cells/well and incubated at 37 °C in a 5% CO₂ incubator for 24 h. Then, different concentrations (10, 20, 40, 60, 80, and 100 μM) of GA, the GAN formulation, and blank niosomal dispersions were added to the respective wells in triplicate and further incubated in a 5% CO₂ incubator at 37 °C for 48 h. After incubation, 20 µL of MTT solution (5 mg/mL in PBS) was added and incubated for three hours in a 5% CO₂ incubator at 37 °C. Then, after removing the medium, DMSO (200 µL) was added to each well and incubated for 5–10 min at 25 °C. Finally, the formazan formation was measured by an ELISA microplate reader (Biotek, Santa Clara, CA, USA) at 595 nm, and the cell viability was calculated using the following equation: