Unknown

The effect of compounds (FGF-401) on cell proliferation is assessed using HuH-7 hepatocellular carcinoma cells obtained from the Japanese Collection of Research Bioresources Cell Bank (Cat# JCRB0403) and cultured in the vendor-recommended medium (DMEM high glucose (Amimed Cat# 1 -26F01 -I), 10% foetal calf serum (Invitrogen Cat# 16140-071 ), 1 mM sodium pyruvate (Amimed Cat# 5-60F00-H), 1x Penicillin/Streptomycin (Amimed Cat# 4-01 F00-H)) at 37°C in a humidified 5% C02 incubator. Specifically, 5000 cells/well were seeded in 96-well tissue culture plates (TPP Cat# 92696) in a total media volume of 100 μΙ/well and increasing compound dilutions or DMSO were added 24 hours thereafter in triplicates. 72 hours after compound addition, cells were fixed by adding 25 [ Uwe\\ of 20% glutaraldehyde (Sigma Aldrich Cat# G400-4) and incubated for 10 minutes at room temperature. Cells were washed three times with H20, 200 [ Uwe\\ and stained with 100 [ Uwe\\ 0.05% methylene blue (ABCR GmbH Cat# AB1 17904) for 10 minutes at room temperature. Cells were washed 3 times with H20, 200 ML/well and then lysed by adding 200 ML/well of 3% HCI (Fluka Cat# 84422) for 30 minutes at room temperature with shaking. Optical density was measured at A650 nm.