Transwell invasion assay

Matrigel (BD Biosciences) was melted at 4°C overnight. On ice, a pre-cooled pipette tip was used to draw 100 µl Matrigel into the pre-cooled 300 µl serum-free medium and was well mixed. Then, 25 µl of the aforementioned-diluted Matrigel was obtained and added to a Transwell plate chamber (BD FALCON; product no. 353097; pore size of the inserts, 8 µm; Corning, Inc.), and placed at 37°C for 30 min to polymerize the Matrigel into a gel. A single cell suspension was prepared with a serum-free medium using a conventional method (22), and the concentration of the cell suspension was 5×10⁵ cells/ml. Then, 100 µl of cell suspension was added to the chamber of the Transwell culture plate and 200 µl of serum-free medium. Subsequently, 500 µl of medium containing 10% FBS was added to the lower chamber of the Transwell culture plate, and cultured in RPMI-1640 medium containing 10% fetal bovine serum (both from Gibco; Thermo Fisher Scientific, Inc.) at 37°C under a volume fraction of 5% CO₂. The Matrigel gel and the cells on the upper surface of the Transwell culture plate were then wiped off. The cells were fixed with 10% methanol at 25°C for 30 min. Crystal violet dye at a concentration of 0.1% was used to stain the cells at 37°C for 1 min. The excess crystal violet dye was rinsed with PBS, and the cells were observed and counted under a light microscope (magnification, ×200).