Cell migration and invasion assays

Transwell analysis with a 24-well Transwell chamber (Corning Life Sciences) was used to assess the effects of CD81 on the migration and invasion capacities of prostate cancer cells. Cells transfected with CD81 siRNA or control vectors (3×10⁴ cells/well) were seeded and incubated in serum-free culture medium in the upper chamber. The lower compartment was filled with 500 µl complete medium containing 10% FBS. For invasion assays, the upper chambers were pre-coated with Matrigel (BD Biosciences). After incubation for 24 h at 37°C with 5% CO₂, the cells remaining on the upper membranes were removed, and migratory or invasive cells on the lower chamber membranes were fixed with 4% paraformaldehyde for 20 min at room temperature and stained with 0.1% crystal violet for 30 min at room temperature. Five random fields from each membrane were counted with a light microscope (magnification, ×200).