cell culture
B16 mouse melanoma cells were obtained from the Culture Col-
lection and Research Center (CCRC, Hsinchu, Taiwan). The cells
were cultured in DMEM medium containing 10% heat-inactivated
foetal bovine serum, 2 mM glutamine, 100 Units/ml penicillin
and 100 lg/ml streptomycin in a humidified 5% CO2/95% air con-
trolled incubator at 37 C. Cell viability was detected with MTT as-
say. MTT is a tetrazolium salt and is converted to insoluble
formazan by mitochondrial dehydrogenase of living cells. Briefly,
after cells were incubated with samples (0.05–1 mg/ml) for 72 h,20 ll MTT (5 mg/ml stock solution) were added to each well. After
1 h incubation, the reaction was terminated by addition of di-
methyl sulfoxide. The optical density of each well was measured
at 570 nm.