HUVEC cell migration and invasion assay

Cell migration assay was performed using a chamber (Corning, Tewksbury. MA) with 8.0-µm polycarbonate filter inserted in 24-well plates as described before (18). HUVECs (4×10⁴ cells/well) in serum-free medium were added to the upper chamber wheras CM was filled in the lower chamber. After cultured at 37 °C for 36 h, the nonmigrated cells were removed from the upper surface of the membrane by scraping with a cotton swab. The migrated cells were fixed with methanol, stained with crystal violet, and photographed under an inverted microscope. Migration was assessed by counting the number of stained cells from 10 random fields at ×200 magnification. The relevant effect of CM was normalized to the total cellular protein