3.2. Tubulogenesis assay (see Note 2)

To assay tube formation, coat 35mm plates with Cultrex BME by thawing out the stock solution (stored at −80 °C) on ice. Once thawed, pipette out 150 μl and coat the plates evenly, cover and allow the material to set in a tissue culture hood at room temperature. Count out and plate early passage HUVEC at a density of about 45,000 cells/cm² and incubate overnight in BSA solution (negative control), FBS positive control, or with a pro- or anti- angiogenic compound of choice (such as VEGF solution and SEMA4D solution).

16 to 20 hours later cells are fixed in 0.5% glutaraldehyde and photographed (Fig. 2).

Endothelial cells were grown in serum-free endothelial growth media with the indicated factors on a substrate of reconstituted basement membrane extract, then fixed and analyzed for tube formation. Media containing 0.1% BSA was used as the negative control (CTRL). Media with BSA and 150 ng/ml bFGF or 10% FBS were the positive controls. Media containing SEMA4D is shown at the indicated dilutions (1).

Quantification of results is determined using Image J, measuring and summing the length of all tubular structures observed in 10 random fields in a phase contrast microscope, for three independent experiments (see Note 9).