Transwell Invasion Assay for Cell Migration

Transwell membranes were coated with Matrigel (40 μg/mL, Corning) at 37 °C for 1 h. The cells treated as indicated were seeded into the precoated upper chambers at a density of 5 × 10⁴ cells/mL in serum-free medium, and the lower chambers were filled with medium containing 20% FBS as the chemoattractant. After culturing for 24 h at 37 °C, the noninvasive cells on the upper surface were removed with a cotton swab, and the cells that migrated to the lower surface through the membrane were fixed with 4% paraformaldehyde for 10 min. The fixed cells were then stained with crystal violet (Beyotime Biotechnology) for 10 min, washed with tap water, and counted under an inverted microscope (Olympus).