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Cell Culture and Determination of Cytotoxicity. Metabolically active H4IIE rat hepatoma cells were grown in Dulbecco¡¯s modified Eagle¡¯s medium (4.5 g/L glucose, 2 mmol/L L-glutamine, 100 units/mL penicillin, 100 µg/mL streptomycin, and 10% fetal bovine serum)in a humidified atmosphere (37 ¡ãC, 5% CO2). The effect of isolated compounds on cell viability was determined using the MTT assay. The cells were plated in 96-multiwell plates with 10 000 cells/well.The cells were allowed to attach for 24 h and then treated with different concentrations of the compounds for 24 h. After this treatment the medium was changed and the cells were incubated for 3 h under cell culture conditions with 0.7 mg/mL MTT. After this incubation the cells were lysed with 50% EtOH/49% H2O/1% HOAc. The concentration of reduced MTT as a marker for cell viability was measured at 560 nm. Data are given as mean SEM of at least three independent experiments. The significance of changes in the test responses was assessed using a one-way ANOVA followed by LSD test (Analyze-it, Leeds, UK); differences were considered significant at P 0.05. |
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