Tubeformationassay

Description
This is a fast and easy assay to test the angiogenic/anti-angiogenic properties of molecules. As compared to other angiogenesis assays, such as chicken chorioallantoic membrane assay, it is convenient and can be performed in any cell culture lab. 

Procedure
1. Day 1: Incubate matrigel on ice at 4 degree C ONT.
2. Transfer 150 ul of pre-cooled matrigel to a 48-well plate (Plate A). Perform this on ice. Allow it to solidify at 37 degree C for 30 min.
3. Collect endothelial cells, count and dilute to 4 x 105 cells/ml in cell culture media. 
4. Dilute the molecules to be tested to required concentration in cell culture media (Make 2x of the final required concentration). Filter this and serially dilute 1:10, 1:100, 1:1000.
5. Transfer 110 ul of each of diluted endothelial cells to respective well of a 96-well plate (Plate B). Add 110 ul of respective diluted molecule (This will make the final concentration to 1x. Mix the cells with the added protein solution by pipetting them up and down three times. Transfer 200 ul of the mixture to the 48-well plate (Plate A). 
6. Incubate at 37 degreeC, 5% CO2 for 3 to 30 h.
7. Day 2 and 3: Observe endothelial tube size and numbers under microscope and photograph each well. Any increase or decrease in the formation of tubes in the test wells as compared to control wells (no test molecule added) will indicate that the molecule is angiogenic or anti-angiogenic, respectively.



Recipes
Matrigel: BD Sciences #354234
Endothelial cells (HUVEC) or tumor cells (3B11)
Angiogenic/anti-angiogenic molecule
cell culture medium specific for the cell line used 


Supplies
Plate: 48 well and 96 well plate.
CO2 incubator