The overall goal of the following experiment is to measure tube formation of various tumor cell types in vitro as a way to gauge vascular activity in vivo. This is achieved by adding tumor cells onto matrigel in order to induce two formation. As a second step.
Cells on matri gel are incubated overnight at 37 degrees Celsius, which allows tube formation to take effect. Next, the tubes produced by each cell type are imaged. In order to quantify the average number of tubes formed by each cell type results are obtained that show the potential capacity of these cells to form vasculature in vivo based on xenograft tumor models.
This method can help answer key questions in the field of cancer research, such as how to measure vascular activity in vitro To prepare tumor cells begin by culturing UAD seven melanoma, B 16 F1 MDA, MB 4 35, or colon HCT one 16 cells. In DMEM supplemented with 10%FPS and penicillin culture. Human microvascular endothelial cells or H ECS in EBM two medium supplemented with one microgram per milliliter hydrocortisone, one nanogram per milliliter, epidermal growth factor 10%FBS and penicillin.
Streptomycin wash the cells with PBS, then detach with 0.05%trypsin EDTA, pellet the cells at 2000 RRP M for five minutes. Then wash them again with PBS. Count the cells using a hemo cytometer to prepare the matra gel warm.
A one milliliter eloqua of growth factor reduced matri gel to room temperature. Then immediately place it on ice for 10 minutes. Pipette 50 microliters into the wells of a 96 well plate.
And to incubate the plate at 37 degrees Celsius for 30 minutes, resuspend one to two times 10 to the fifth tumor or endothelial cells in DMEM or e BM two respectively and load them onto the top of the matrigel. If studying the effects of reagents on tubule development, add the reagent to the serum free medium incubate overnight at 37 degrees Celsius. For image analysis, add 10 microliters of 10%formal and saline based solution for 10 minutes.
Then image using a phase contrast microscope under a 10 x objective. Count the number of tubules in three to five random fields from each. Well then take an average here.
HMCs were used as a positive control as these tubules developed from clear elongated cell bodies that connected to form A polygon network. B 16 F1 U 87 and MDA MB 4 35 cells developed vascular tubules similar to those formed by H ecs. But HCT one 16 cells did not.
Cell dose dependent tube formation was tested in U 87 cells. As demonstrated in this figure, 10, 000 cells formed discontinued tubules once the cells were doubled. A solid vascular network was comparable to that seen in H ecs.
In contrast, in inaugurating with less than 5, 000 cells failed to form a vascular phenotype. After watching this video, you should have a good understanding of how to successfully set up a matrigel tube assay experiment to measure the tube formation of cancer cells in vitro.
