Transwell Migration and Invasion Assay

The migration and invasion of cells were examined using a Transwell assay. For cell migration assay, a total of 2 × 10⁵ cells were diluted using FBS-free medium and placed onto the upper chamber (BD Biosciences). For cell invasion assay, cells were added into the upper chamber which was precoated with Matrigel (Sigma-Aldrich). Then, 500 µL of culture medium containing 10% FBS was added into the lower chamber. Following overnight incubation, nonmigratory/-invasive cells were removed using a cotton swab. The migrated/invaded cells in the lower chamber were fixed using 4% paraformaldehyde and stained with 0.5% crystal violet. The numbers of cells were counted within 5 randomly selected fields by an inverted light microscope (magnification ×200, Olympus Corporation).