3D Angiogenesis Assay with BMP9 and VEGF

PAECs were cultured in a collagen and fibronectin gel matrix as previously described^{50 (link)}. Briefly, cells were combined with the gel at a final concentration of 1×10⁶. The suspension was then pipetted into an angiogenesis μ-slide (Ibidi, Germany) in a volume of 10 μl/well. After 10 minutes at 37°C to allow polymerization, the gel was overlaid with 40μl of media (EBM-2, Lonza and 2% FBS), with or without 5 ng/ml BMP9 or 30 ng/mL VEGF₁₆₅ (both R&D Systems) individually or combined. The 3-D cultures were left at 37°C for 24 hours and tube formation confirmed by brightfield microscopy. Images were collected and parameters measured and quantified using Image J software. In order to gain confocal images, gels were first fixed in 4% PFA, washed in PBS and incubated with Rhodamine labelled ULEX Europaeus Agglutinin1 (Vector Laboratories, Burlingame, CA) overnight at 4°C. After 3× 10 minute PBS washes, nuclear staining was carried out using 10μl of VectorSHIELD mounting media with DAPI (Vector Laboratories) for 30 minutes. A final x3 wash in PBS was completed.