Fibrin gel bead sprouting assay

A previously established fibrin gel bead sprouting assay was optimized to study angiogenesis (Nakatsu et al., 2007; Nakatsu and Hughes, 2008). A total of 2500 Cytodex microcarrier beads (Sigma-Aldrich, C3275) were coated with 1 × 10⁶ EA.hy926 in medium at 37°C and 5% CO₂. The tube was shaken gently every 20 min for 4 h. The coated beads were then transferred to a dish containing 5 ml EGM2 medium and left overnight. The next day, the EA.hy926-coated beads were resuspended at a concentration of 500 beads/ml in 2 mg/ml fibrinogen (Sigma-Aldrich, F3879) solution containing 0.15 U/ml aprotinin (Meilunbio, MB3095). Then, 0.625 U/ml thrombin (Meilunbio, MB1368) was added to a 24-well plate, followed by the addition of the fibrinogen/bead solution. The plate was left at room temperature for 5 min and then placed in an incubator at 37°C and 5% CO₂ for 15 min to generate a clot. Finally, fibroblast NIH-3T3 cells were seeded on top of the fibrin gel at a concentration of 2 × 10⁴ cells/well in 1 ml EGM2 medium. The medium was changed every other day. Sprouting images were captured after 2–4 days.