Transwell chambers (24-well; Corning, Inc.) with a 5-µm pore filter were coated with a mixture of 60 ml 1:9 Matrix (BD Biosciences) and serum-free RPMI-1640 medium to form a matrix barrier. Precoating was performed at 37°C for 1 h and the Transwell inserts were refrigerated at 4°C overnight. Before inoculation, RPMI-1640 medium was used to hydrate the basement membrane for 30 min at 37°C. The upper chamber cells were filled with 200 µl serum-free RPMI-1640 medium, and the lower chamber was filled with 500 µl RPMI-1640 medium supplemented with 10% FBS. Following incubation at 37°C for 48 h, the cells were washed twice with PBS (5 min per wash) and fixed with 600 µl polyformaldehyde for 15 min at 4°C. The non-invading cells were wiped with a cotton swab, washed with ddH2O three times and stained with 5% crystal violet solution for 5 min at room temperature (23 (link)). The number of invading cells was counted in four random fields using an inverted phase contrast microscope (Olympus Corporation) at a ×50 magnification.