Transwell migration and invasion assay

Cell migration was also studied using Corning Transwell polycarbonate membrane inserts with 24 pores (pore size 8.0 μm, membrane diameter 6.5 mm). For cell invasion assay, we used the same protocol, except that the Transwell inserts were coated with a layer of basement membrane extract to mimic extracellular matrix. HCC cells in the logarithmic growth phase were starved in serum-free media 24 hours prior to assay. After incubating cells for 24 hours in serum-free media, cells were harvested and counted. The cells were washed with sterile PBS and suspended at 1×10⁶/ml in serum-free medium. 100 μl cells were added into the upper chamber and 650 μl DMEM medium with 10% FBS and other reagents was used as a chemoattractant in the lower chamber. Cell migration and invasion was allowed to progress for 24h at 37°C with 5% CO₂. Afterward, the matrix gel and cells on the top membrane surface were removed with a cotton swab. Transwell membranes were then stained with 0.1% crystal violet and light microscopy was used to photograph the cells that migrated through the membrane. The number of migrating cells per field was counted under microscopy.