8 m pore membranes

For migration assays, 4 × 10⁴ HCC cells were resuspended in serum-free medium and then seeded onto the upper chamber of 8-µm pore membranes (Corning, USA). Complete medium containing 10% FBS (Gibco, Australia) as a chemoattractant was added to the lower chamber. Twenty-four hours later, the cells located on the lower chamber were fixed with 4% paraformaldehyde, stained with 0.2% crystal violet and photographed using inverted fluorescence microscope. Five fields per membrane were randomly selected and then the average number was determined. For invasion assays, 100 μl of Matrigel (200 mg/ml) were added to the upper chamber at 37 °C for 6 h, followed by the implant of 8 × 10⁴ HCC cells. Other experimental procedures were performed as previously described. Triplicate assays were used for each experiment.

The cell migration assay was performed using modified Boyden chambers in 24-well dishes with Transwell filter inserts provided with 8 µm pore membranes (Corning Inc., Corning, NY, USA). Aliquots of 4 × 10⁴ cells were seeded into each upper chamber of the insert in serum-free medium, and complete medium was added to the lower chamber. After 12 or 24 h, cells were fixed with 4% paraformaldehyde and stained using 0.1% crystal violet. Cells in the upper chamber were carefully removed, and the cells that migrated through the lower side of the filter were imaged (Olympus IX81) and quantified with ImageJ.