Evos fl auto cell imaging system evos fl

Transwell inserts (Costar, Corning, USA) with 8-μm polycarbonate membranes were used to assess the migration ability of cancer cells. The upper chambers coated with Matrigel (Corning, USA) were used for the invasion assay in vitro. Briefly, 200 μl serum-free medium containing 1×10⁵ cells was added to the upper chambers. The bottom chambers received 600 μl culture medium containing 10% FBS. After incubating for 1 day (Huh-7) or 2 days (Hep 3B), the cells remaining in the upper chambers were gently wiped with wet cotton swabs. The migrated or invaded cells were fixed with methanol and stained with 0.1% crystal violet for 15 minutes. Five fields were randomly selected for imaging using the microscope EVOS FL Auto Cell Imaging System (EVOS FL, Thermo Fisher Scientific, USA).

The optical density was monitored at a wavelength of 750 nm using a UV-VIS spectrophotometer (Hach Lange DR6000). Cell concentration and cell size were measured in triplicate with the Beckman Coulter Multisizer III (Beckman Coulter Inc., USA) using a 50 µm aperture tube. Microscopic analysis was performed with the microscope EVOS FL Auto Cell Imaging System (EVOS FL, ThermoFisher Scientific). All samples were diluted 200 times in Isotone^® II diluent solution before measurements. Dry weight concentration was determined by filtrating 10 ml of culture broth on a pre-dried and pre-weight 55 mm Whatman glass fibre filter paper (GF/F; Whatman International Ltd, Maidstone, UK). The filter was washed with filtered demineralized water and subsequently dried overnight at 100 °C before weighing. The dry weight of the samples was calculated from the difference in weight between the dry filters with and without biomass.