Transwell Migration and Invasion Assays

Transwell migration assays were performed in a 24-well Boyden chamber (6.5 mm diameter, 8.0 μm; BD) according to the manufacturer’s instructions as previously described [3 (link)]. In brief, 5 × 10⁴ cells/well in serum-free medium were seeded in the upper chamber, and the lower chamber was filled with complete media. After 24 h, cells in the upper chamber were gently removed. Migrated cells on the lower side of the membrane were fixed with methanol, stained with 0.1% crystal violet, and then counted at 40× magnification in five random fields per well. For the in vitro invasion assay, experiments were performed with Matrigel matrix-coated Transwell chambers.