Transwell invasion and Matrigel assays

Cell invasion analysis was performed using a Matrigel-coated Transwell assay. Matrigel was defrosted from −20°C to 4°C in a refrigerator overnight and the experiment was performed on ice at all times, with all consumables pre-cooled. A total volume of 100 µl diluted Matrigel in serum free-cold DMEM was added to the upper chamber of the Transwell, which was subsequently inserted into 24-well plates. After incubating the Transwell at 37°C for 4 h, gelled Matrigel was gently washed with warmed serum-free RPMI-1640 medium. After 24 h of transfection, the cells were trypsinized and re-suspended in serum-free 1640 medium at a density of 1×10⁵ cells/ml; 100 µl of this cell suspension (1×10⁴ cells) was plated in the upper chamber and 600 µl 1640 medium containing 10% FBS was plated in the lower chamber. After 24 h of incubation at 37°C, the non-invasive cells remaining in the upper chamber of the Transwell plate were scraped off with a cotton swab. The invading cells on the lower surface of the chamber were fixed with 4% formaldehyde for 15 min at room temperature and subsequently stained with 0.1% crystal violet for 5 min. After washing with PBS, invasive cells were counted using a light microscope (magnification, ×100).

Cellular migration was also detected by a Transwell assay and followed the same protocol as the Matrigel invasion assay, with the exception that Matrigel was not used to coat the Transwell plates.