Protocol for Aortic Ring Assay

Procedures

1. Cover a 48-well plate with Matrigel (100μl/well) of and incubate for 30 min at 37°C, 5% CO2.
2. Sacrifice the1-2 month old mice/rats (WT/mutant) and excise thoracic aorta. Transfer the aorta into a dish with sterilized 1x PBS buffer. After removing the fibroadipose tissue, section arteries into 1-1.5 mm long cross sections, rinse the sections five times with endothelial cell growth medium (ECGM), and place them on the Matrigel-coated wells.
3. Cover the artery rings with an additional 100μl of Matrigel. If there are some bubbles, use pipette to remove them. Incubate the rings in 1.5 ml of ECGM medium.
4. After 24 hour incubation, replace the medium with 1.5 ml of endothelial cell growth medium (or containing with the interesting peptides/compounds/others).
5. After 2 days, the medium was replaced with medium of the exact composition as described in step 4.
6. At the day 4, take images of aortic rings. Delineate the outgrowth area and measure the outgrowth micro-vessel with the Image Pro Plus software (Media Cybernetic).