Cells were starved in 0.1% serum for 24 h, detached by 2mM EDTA/PBS, and 80,000 cells were seeded in serum free medium on 24-Transwell membranes (8 μm pores). For invasion assay, the upper side of the membrane was coated with 70 μl of 1 mg/ml Matrigel. The lower chamber was filled with medium, supplement with 0.1% FBS with or without 50 ng/ml HGF. After 48 h, membranes were fixed in 1% glutaraldehyde and stained with 0.5% crystal violet. Membranes were punched out with a 6 mm diameter dermal biopsy punch and crystal violet was solubilized in 10% acetic acid and measured. Percent of migrated cells was calculated as: absorbance 595 nm from migrated cells / absorbance 595 nm from migrated + nonmigrated cells.
