Matrigel was diluted 1:3 in serum-free medium before being added to the upper chamber of a 24-well Transwell plate (Millipore, Billerica, Massachusetts, USA). HCC cells were trypsinized and counted manually under a microscope. A cell suspension of 50,000 cells/mL in serum-free medium was prepared and 100 μL of the suspension was loaded into the top chamber. The bottom chambers were filled with medium with 10% FBS. Invasion was halted after 24 hrs incubation in a 37°C incubator (5% CO2) by scraping the non-migrated cells off the upper chamber using a cotton swab. After being fixed in 4% paraformaldehyde, migrated cells were stained with 0.05% Crystal Violet. Migrated HCC cells were counted under an inverted light microscope.
