HUVEC Proliferation Assay

Human umbilical cord vein endothelial cells (HUVEC) were purchased from ATCC and cultured in EGM-2 media (Lonza) in a humidified incubator at 37°C and 5% CO₂. HUVEC were seeded at a density of 5000 cells/ well in a 96-well plate. After 24 hours, the EGM-2 was removed, the cells rinsed twice with PBS and M199 media supplemented with 1% FBS. Cells were then treated with one of the following: (1) unconjugated bFGF in M199 media with 1% FBS; (2) mvbFGF 10:1 in M199 media with 1% FBS; and, (3) mvbFGF 30:1 in M199 media with 1% FBS. For each treatment (conjugated and unconjugated), a range of bFGF concentrations were tested over a range that was used to assess its bioactivity using HUVECs in previous studies (0, 5, 10, 25, 50, and 100 ng/mL).³⁵ After 72 hours, the media was aspirated, the plate rinsed with PBS and frozen for analysis of cell proliferation with the CyQUANT assay kit (Invitrogen), following the manufacturer’s instructions. The data was normalized to the cell number in the control well after 72 hours (no bFGF or mvbFGF added).