Tgfb2

Left ventricular explant culture was performed and adapted as previously described.^{8 (link)} Hearts were isolated in cold PBS from E12.5 Bmx^CreER;R26^fsGFP embryos which were tamoxifen induced at E11.5. After removal of the atrium, the right ventricle, and septum of the heart, the left ventricle was rinsed with PBS twice to remove circulating cells, placed on the surface of pre-casted basement membrane matrix (200 μl/well) (BD Matrigel, growth factor reduced) in Nunc 4-well plates, and then sandwiched by adding an additional 150 μl of Matrigel. The Matrigel was prepared by addition of an equal volume of Endothelial Cell Growth Basal Medium (R&D, Cat# CCM029) with or without a testing growth factor, including Vegf120 (R&D, Cat# 494-VE-025) and Tgfb2 (R&D, Cat# 7346-B2–005). The final concentration of Vegf120 growth factor in the cultures was 10 ng/ml and Tgfb2 growth factor in the cultures was 2 ng/ml or 10 ng/ml, respectively. Heart explants were cultured at 37°C for up to nine days. The features of angiogenesis including transmural migration, sprouting, and endothelial networking were examined and photographed.