The surface of the Transwell membrane (Corning Inc.) was coated with a 1:6 dilution Matrigel solution 60 µl (BD Biosciences). The Transwell was incubated with Matrigel for 30 min at 37 °C for gelling. Cell suspensions were prepared at cell densities of 5 × 105 per ml. Cells were starved with culture medium containing 2% FBS for 8 h. Then 100 µl of cell suspension was added to each Transwell insert and 600 µl of 20% FBS-containing culture medium was added to the lower chamber of plate and incubated for 24 h at 37 °C. Cells on the lower surface of the membrane (migrated cells) were fixed in methanol and stained with 0.1% crystal violet. The number of migrated cells was counted from five random fields of vision at 200× magnification, and averaged to obtain the final count. All experiments were performed in triplicate.