HUVEC Migration Assay

Wells of a 12-well plate were coated with 0.2% gelatin. HUVECs were seeded at a density of 150,000 cells/well 24 hours prior to starting the assay. Using a 1 ml pipette tip, crosses were scratched into the confluent layer of HUVECs. The wells were then washed with excess PBS to remove cell debris and media and replaced with M199 with 1% FBS, containing either (1) unconjugated bFGF, (2) conjugated bFGF 10:1, or (3) conjugated bFGF 30:1, all at a concentration of 50 ng/ml. Scratches were imaged at 0 and 24 hours post scratch and the area without cells was quantified using ImageJ. The percent open wound area was calculated by comparing the open scratch area at 24 hours to the open scratch area at 0 hours. The change in scratch area between 0 and 24 hours for each treatment group was compared with untreated scratches.