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4. Cell culture conditions; WM266-4 (ATCC Number: CRL-1675), SK-MEL-28 (ATCC Number: HTB-72) and CHL-1 cells (ATCC Number: CRL-9446) were cultured in DMEM (Dulbecco's Modified Eagle's Medium) containing 10% fetal calf serum and 30 μg/ml of penicillin and 42 μg/ml of streptomycin in 5% CO2 in an incubator at 37 °C. A Cell-Based Assay conditions; WM266-4, SK-MEL-28 and CHL-1 cells were seeded on 96-well microplates (1.0 × 104 cells per well), respectively. Test compounds were dissolved in methanol at appropriate concentrations and were treated for 72 h at 37 °C in a 5% CO2 atmosphere. Cell proliferation assays were carried out by the WST-8™ (Nacalai Tesque, Kyoto, Japan) protocol. The absorbance (A450) of each well was measured by a Wallac 1420 multilabel counter (Amersham Biosciences, Piscataway, NJ).
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