Transwell Migration Assay

To assess the level of cellular migration, cells in the logarithmic growth phase were routinely harvested by digestion and centrifugation. The cells were resuspended in serum-free medium, and adjusted to 1 × 10⁵/ml. Then, 200 μl of each suspension was added to the upper Transwell chamber, whereas 500 μl medium containing serum was added to the lower chamber. After 24 h, the non-migrated cells on the upper surface of the filters were wiped off using a cotton swab. Migratory cells on the lower membrane surface were fixed with 4% paraformaldehyde for 15 min, stained with 1% crystal violet for 20 min, washed with single distilled water, and dried. Cells were then visualized with an OLYMPUS IX71 Inverted Microscope and the number of migratory cells was counted. Data were presented as the means ± standard deviations of the results from three independent experiments.