Boyden chamber-based cell migration assay

Migration assay was performed in 24-well cell culture inserts (Falcon) with 8 μm pore size membrane. P and S1 cells were trypsinized from 100 mm plates, then washed with a serum-free DMEM twice. The cells were seeded into the inserts in the serum-free medium with or without 100 ng/mL hepatocyte growth factor (HGF, Gibco), whereas the lower chambers of the wells were filled by the medium containing 10 % FBS. After 24 hr incubation at 37°C, the cells on the top side of the insert membranes were removed by cotton swabs. The cells migrated to the bottom side of the insert membrane were fixed with ice-cold methanol at −20°C for 10 min, then stained with 0.5% crystal violet (Farco Chemical Supplies) in 20% methanol at room temperature for 10 min. Migrated cells were counted and captured using light microscopy.