Matrigel capillary tube formation: Matrigel was purchased from Becton-Dickinson (Bedford, MA, USA), diluted to 4 mg/ml with cold phosphate-buffered saline (PBS), and added to 24-well plates to a total volume of 400 μl in each well. Plates were stood at 37℃ for 1 hour to form a gel layer. After gel formation, a total of 2×10（5）/ml HUVEC cells were applied to each well and incubated with the supernatants from cell cultures for 24 hours or untreated group; VEGF 165 or (10 ng/ml) group; control (1640 medium instead of the supernatants from cell culture). Plates were incubated at 37℃ for 24 hours with 5% CO2 , then viewed (original magnification ×40) and photographed using an microscope. (. At least 5 fields were examined per well, and each experimental condition was tested in triplicate.

Good Luck.

Cynthia

How do you make your control? How is your negative control? On a similar assays I used basal EGM-2 with no added growth factors and only 0.1% FBS as negative control [in which I also starved the cells for 3h before the assays.] The positive control was cells in complete EGM-2 .

I added VEGF directly on the gels and resuspended the cells in basal EGM-2/0.1%FBS for my experiments [as I only wanted to see the effect of controlled concentrations of VEGF which was in the range of 1nM ].

You should see better structure formation with VEGF compared to the negative control but not necessarily better structure formation than in your positive control which is the complete EGM.

I must add that a cell batch I once ordered in did not respond to VEGF and I never found out why.