4.3. A Cell Proliferation/Cytotoxicity Assay

The rate of inhibition of cell proliferation was measured by two methods including direct cell counting and an MTT assay. B16F10 cells were cultured in a 24-well plate (6 × 10⁴ cells/well) for direct cell counting and in a 96-well plate (1 × 10⁴ cells/well) for the MTT assay and were treated with either vehicle or various concentrations of carvone for 48 h. The cells in the 24-well plate were trypsinized, resuspended in the growth medium, and stained with trypan blue (Invitrogen, Carlsbad, CA, USA). Viable cell numbers were determined using an automated cell counter, CELLOP^® (Small Machines Company, Seoul, Korea). Additionally, for the evaluation of cell cytotoxicity, the number of nonviable cells was also counted and the percentage of dead cells was calculated (CELLOP^®). For the MTT assay, the cells were incubated with 60 µL of the MTT reagent (4 mg/mL in PBS) for 3 h at 37 °C to allow formazan crystals to form. After that, the medium was removed and 300 µL of DMSO was added into each well to dissolve the formazan crystals. The colored solution in each well was analyzed at 595 nm on the microplate reader. Furthermore, to determine whether the proliferation of Hs68 and HaCaT cells was also affected by carvone treatment the same method was employed as above.