Transwell migration and invasion assays

Cell migration and invasion detection was performed using Transwell chambers (EMD Millipore). The transfected C33A and SiHa cells were resuspended in serum-free medium. For the invasion assay, 1×10⁵ cells in 100 µl serum-free medium were seeded into the top chamber coated with Matrigel (BD Biosciences). Complete medium (500 µl) containing 10% FBS was added to the bottom wells. After incubating for 12 h at 37°C, the non-invaded cells in the upper chamber were removed with a cotton swab. Subsequently, the invaded cells were stained with 0.1% crystal violet at room temperature for 10 min. Five fields per filter were counted using a light microscope (Nikon TE2000; Nikon Corporation) (magnification, ×40). For the migration assay, 5×10³ cells were added to the top chambers. Except for the Matrigel coating, the migration assay was the same as the invasion assay. Matrigel (10 µl) (1:8) was added to the upper chamber for 4 h at 37°C.