2.4. Matrigel® invasion assay

Twenty-four Transwell® permeable plate supports with 8.0 μm polycarbonate membrane and Matrigel® were purchased from Corning Life Sciences (Tewksbury, MA). A concentration of 5mg/ml of Matrigel was used for coating supports. Matrigel® invasion assay was performed as published previously [35]. Briefly, cells were seeded in 6 well plates, washed one time with 1X PBS and treated with either SB505124 (20 µM) or 0.5 % DMSO (diluted with 0.9% saline) for 24 hours. The medium was aspirated, and cells were washed once with 1X PBS, detached using sterile 20 mM EDTA in PBS and washed once with 0.9% saline. Cells were re-suspended in serum-free medium with/without treatment based on the final treatment. Using Countess automated cell counter (Invitrogen), 100,000 cells were seeded on to the Matrigel® in the upper chamber of the transwell plates filled with 100 µl of serum-free medium. Cells that invaded the matrigel and reached the bottom layers of the supports after 24 hours incubation were fixed using 3.7 % paraformaldehyde then stained with 0.5% crystal violet solution. Three bright field images of each insert were taken using an inverted microscope and three blinded reviewers counted the invaded cells. The average number of invaded cells from every three images was calculated and considered for the analysis.