Cell invasion assay

Cell invasion potential was measured with a Boyden transwell chamber consisting of upper inserts with 8-μm-pore-size filter membranes at the bottom of the inserts and lower wells in 24-well cell culture plates (Corning Life Sciences). Add 20 μl of 1:6 diluted Matrigel (2–3 mg/ml protein) to the center of each cell well inserts. Place coated inserts in incubator to allow the Matrigel to solidify for 20–30 min. Cells (3.5 × 10⁵ cells in 0.2 mL) suspended in serum-free medium with 0.1% bovine serum albumin were seeded into the inserts of the chambers. The inserts were then placed over the wells filled with 0.5 mL 10% FBS culture medium and incubated in a 37°C incubator for 16 h. Cells that had not penetrated the filter membrane in the inserts were wiped off with cotton swabs, and the cells on the underside of the filter membrane were fixed and stained with the HEMA-3 kit (Fisher Diagnostics). Invaded cells were counted in total 10 fields for each sample under microscope with 10X objective and stained cell number per field was calculated [34].