Transwell migration and invasion assays

Migration and invasion assays were performed in 24-well plates with inserts (8 μmol/L pore size, Corning) without or with Matrigel. Cancer cells (2 × 10⁴ cells/well) were added into the upper chambers in serum-free media. Meanwhile, RPMI 1640 containing 5% FBS was added to the lower chambers. After incubation at 37 °C in 5% CO₂ for 24 h (migration assay) or 36 h (invasion assay), the upper chamber was cleaned with a cotton swab and the lower chamber was fixed with 4% paraformaldehyde, dyed with 0.1% crystal violet, and then washed with water for three times. An inversion microscope (Olympus CKX53, Japan) was used to image the cells under a microscope. The number of cells per field was calculated using the Image J Version 1.48 software (National Institutes of Health, Bethesda, MD).