Transwell Assay for Cell Invasion

Transwell assay was conducted, as described previously [16 (link)]. Briefly, serum-free cell suspensions (2.5 × 10⁴ cells/mL) were made and 0.1 mL of the cell suspension was seeded to the top chamber of the transwell plates. Culture medium containing 10% FBS was added into the lower chamber. Cells were cultivated for 24 h at 37°C 5% CO₂. Membranes were cleaned using a cotton swab, followed by fixing with 4% polyformaldehyde (10 min). After washing twice, the top chamber was stained with 0.5% crystal violet (Sigma-Aldrich; St. Louis, USA) for 15 min at room temperature. The experiments were performed in triplicate. Cells were observed and counted under Olympus CX43 light microscope (×40 magnification). Then invasive cells in three fields were counted, and the average number was calculated.