2.7. Cell Migration and Invasion Assay

The upper chamber of the basal membrane was coated with 5 μg/μL Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). Transwell filters (pore size, 8 μm, Corning Incorporated, Corning, NY, USA) were placed in 24-well plates. At 24 h after transfection, cells were added to the compartment of a transwell chamber containing DMEM with 0.1% bovine serum albumin. DMEM containing 10% fetal bovine serum was used as the chemoattractant. After 24 h at 37°C, cells on the upper membrane surface of the filter were gently wiped off with a cotton swab, and the filters were fixed with 95% ethanol for 30 minutes and then stained with 0.2% crystal violet solution for 30 minutes. Invasion cells adhering to the undersurface were fixed and stained with 0.1% crystal violet. The total number of invading cells was counted in five representative fields under microscopy (200x magnification).