Mouse Aortic Ring Assay

In Vitro Mouse Aortic Ring Culture Protocol

1.Euthanize adult mice aged approximately 8 weeks. Carefully dissect the thoracic aorta using sterilized surgical instruments and wash it twice with sterile PBS.

2. Place the cleaned thoracic aorta in a 35mm dish containing Opti-MEM medium. Carefully cut it into 0.5mm circular rings using ophthalmic scissors.

3. Transfer the aortic ring into 1ml Opti-MEM medium in a 24-well plate and incubate overnight in a 37°C incubator.

4. The following day, add 20 μl of growth factor-reduced matrix to each well of the 24-well plate. Incubate at 37°C for 10-15 minutes. Once the matrix solidifies, place an aortic ring on the solidified matrix, add 20 μl of matrix to each ring, and incubate the plate in a 37°C incubator for 10-15 minutes.

5. Add Opti-MEM medium containing 2.5% fetal bovine serum, 30 ng/mL VEGF, and 1% antibiotics to the well plate. Administer the desired treatment and incubate in a 37°C incubator.

6. After 2-3 days, depending on the growth of the aortic ring, replace the culture medium once and continue incubating at 37°C.

7. On the 5th day of cultivation, use a Nikon TI2-U inverted microscope to capture images, and analyze vascular sprouting using ImageJ software.