Ulex europaeus lectin

Early passage HUVECs were coated on cytodex-3 beads (GE Healthcare) at a density of 10 million cells/40 μl beads and incubated in suspension for 3–4 hours with gentle mixing every hour. They were plated on TC treated 6 well dishes overnight and resuspended in a 2 mg/ml fibrin gel with 200,000 human smooth muscle cells. The gel was allowed to polymerize and complete EGM-2 media was added. Sprouts were visualized from days 3–4 via confocal imaging after overnight incubation with FITC labeled Ulex europaeus lectin (Vector labs). Immunofluorescence imaging was performed on a Yokogawa CSU-W1 spinning disk confocal microscope with 20 0.45 Plan Fluor objective (Nikon).

HUVECs transfected for 24 h and were coated on cytodex-3 (x) beads at a density of 1 million cells per 25 uL beads and incubated in suspension for 3–4 h with gentle mixing every hour. In some cases, TPI was added during the incubation. Beads were then plated on TC treated 24 well dishes overnight and resuspended in 2 mg/ml fibrin gel with 200,000 fibroblasts. The gel was allowed to polymerize and complete EGM-2 media was added. Sprouts were visualized on day 4 via confocal imaging following a 4-h incubated with 1:200 flourescein isothiocyanate (FITC)-labelled Ulex europaeus lectin (Vector labs) [44 ].

Early-passage HUVECs were coated on cytodex-3 beads (GE Healthcare) at a density of 10 million cells/40 μl beads and incubated in suspension for 3–4 h with gentle mixing every hour. They were plated on Tissue Culture (TC)-treated six-well dishes overnight and resuspended in a 2 mg/ml fibrin gel with 200 000 human smooth muscle cells. The gel was allowed to polymerize and complete EGM-2 media was added. Sprouts were visualized from days 3 to 4 via confocal imaging after overnight incubation with fluorescein isothiocyanate-labeled Ulex europaeus lectin (Vector labs). Immunofluorescence imaging was performed on a Yokogawa CSU-W1 spinning disk confocal microscope with ×20, 0.45 Plan Fluor objective (Nikon).