8 μm pore polycarbonate membrane inserts

8-μm pore polycarbonate membrane inserts (Becton Dickinson, Franklin Lakes, NJ, USA) were used to measure the HCC cells’ invasive and migration abilities according to the manufacturer’ s protocol. In short, 2 × 10⁶ cells in 250 μL serum free medium were seeded into the upper chamber and 500 μL medium containing 10% FBS was added to the lower chamber. After 48 h in culture, cells on the upper side were removed by a swab, fixed in 100% methanol for 15 min at room temperature, and then stained by crystal violet. Photographs of five random fields under 200 × magnification were captured for quantification analysis with the double-blind method. Three identical replicates were performed and eventually got a mean values.

The invasion assay was performed as reported previously with some modifications [45 (link)]. The cells' invasive and migration abilities were examined using a 24-well trans-well with 8-μm pore polycarbonate membrane inserts (Becton Dickinson, Franklin Lakes, NJ, USA) according to the manufacturer's protocol. 2× 10⁶ cells were planted on the upper chamber. After 48h in culture, cells that appeared on the lower surface of the filter were fixed with 100% methanol and stained with crystal violet and counted in five random ×200 fields using an inverted microscope and with the double-blind method. The experiments were performed in triplicate and got means were calculated.