Mouse Endothelial Tube Formation Assay (In Vitro Angiogenesis)

Preparing Matrigel:

• Thaw reduced growth factor matrigel (BD: 354234) by submerging the bottle on ice and store the matrigel at 4 °C overnight.

Preparing Cells:

·       Wash 90-95% confluent endothelial cells with sterile PBS (1X) without calcium and magnesium twice.

·       Incubate cells (on T25 or T75flask) with warm 0.25% Trypsin-EDTA solution (Cell Biologics, Catalog No. 6914) for 3-5 minutes at 37°C. Once cells detach (slightly taps on the flask can be used to speed up the detachment), add 6-10 ml culture medium (Cell Biologics' Cell Culture Medium supplemented with 5-10 % FBS) to the flask to end the digestion (neutralizing the Trypsin).

·       Filter the cell suspension through a sterile 40µm nylon mesh filter (BD 352340) to remove remaining clumps.

·       Count cells and resuspend cells to a final concentration of 2.5-5.0 x 10⁵ cells/ml in endothelial cell culture medium with 50 ng/ml VEGF.

Tube Formation Assay

·       Pipet 200 µl matrigel into each well of a 24-well plate. Avoid creating bubbles.

·       Incubate the 24-well plate at 37 °C and 5% CO₂ for 30 min to solidify matrigel.

·       Once matrigel has set, seed 50,000 cells -100,000(200ul) into each well.

·       Observe the tube formation after 4-6 hrs. Peak tube formation may occur between 12hrs and 24hrs. 

·       Take image by 4X lenses microscope. 

Note: Adjust the volume of endothelial culture medium and number of cells plated depending on the type of endothelial cell used.