Tube-formation Assay is an in vitro test to study the molecular mechanisms underlying several steps that lead to the formation of new blood vessels.This assay allows to identify compounds and signaling cascades associated to angiogenesis.With one test, we're able to show that a natural compound like RGWE reduces the ability of endothelial cells to form tube-like structure, do not affect cell viability, and that this effect depends on the activation of the MEK/ERK pathway.It is important to perform the test with the right number of cells.In fact, too few or too many cells could not allow the right formation of tubes.For this reason, it is advisable to perform a preliminary test to find out the correct number of the cells.Luca Olga Pastorino, PhD student in my lab, will show the procedures.First, collect Ruta graveolens leaves during the spring and summer months.Put 250 grams of chopped leaves in a conical flask, and add one liter of distilled water, boil, and filter, as described in the manuscript.The next day, use a lyophilizer to lyophilize the leaf extract.After obtaining the powder, weigh it, and divide it into aliquots.Culture the HUVECs in endothelial cell growth medium, according to the text protocol.When the cells reach 80%confluency, passage the cells at a ratio of one to three.Use the cells obtained between passage two and passage five.Once the passaged HUVECs reach 70%confluency, transvect them with one microgram of the vector containing the gene of interest.Combine one microgram of DNA with three microliters of diluted Lipofectamine 2000.Then, remove the medium from the HUVECs, and replace it with one milliliter of fresh complete medium.Add the transvecting solution to the cells, and incubate the cells at 37 degrees Celsius, and five percent carbon dioxide.Three hours after transvection, add seven milliliters of fresh complete medium, and incubate the cells for an additional 24 to 48 hours.Immediately before basement preparation, put a pre-chilled, 96-well plate on ice, and add 50-microliters of cold matrix to each well.While the plate incubates, wash the HUVECs with one milliliter of PBS/EDTA solution.Then, add one-milliliter of Trypsin-EDTA solution to the cells, and incubate for five minutes at 37 degrees Celsius.Collect the medium containing the suspended cells, and harvest the cells via centrifugation.Then, resuspend the cells in one milliliter of PBS.Transfer 0.2-milliliters of the cell suspension to 0.5-milliliters of PBS with trypan blue solution.After holding the suspension at room temperature for five minutes, count the cells in a B
