The overall goal of this procedure is to determine genes or pathways involved in angiogenesis through the use of a rapid and quantitative in vitro method. This is accomplished by first serum starving primary or immortalized endothelial cells the day prior to the assay. The second step is to incubate endothelial cells with calcine for 30 minutes, followed by washing the cells with PBS.
Next, the endothelial cells are tryps inized filter to remove clumps and counted. The final step is to combine endothelial cells with conditioned media containing possible angiogenic factors and dispense them over solidified basement membrane extract. Ultimately, light or fluorescent microscopy is used to visualize tube networks, which can then be quantified using specialized computer programs.
The main advantage of this technique over other methods for assay angiogenesis is that it is easy to set up, relatively inexpensive, produces tubules within hours and is quantifiable demonstrating this technique. Is Erica Bullwinkle, a graduate student in the biology department at American University? Using the appropriate media transfer three B 11 cells into a new flask two days before starting the assay for the best results, the cells should be between the second and sixth passages when used.
Next, grow the cells for 24 hours in supplemented dmm. When the cells are ready, they are serum starved. By replacing the media with reduced serum media, grow the serum starved cells for an additional 24 hours.
For the best results, it is important to confirm with the manufacturer that the concentration of the BME stock solution is 10 milligrams per milliliter. Defrost the reduced growth factor BME in the refrigerator. Since BME solidifies when warm, it is kept cold prior to use.
Next thaw the media needed for the assay for visualization with fluorescence or confocal microscopy add calcine AM to the media at a final concentration of two micrograms per milliliter placed a calcine am labeled cells in the dark for about 30 to 45 minutes. After this time, remove the media and wash the three B 11 cells in DPBS. Next trypsin is the cells by adding one milliliter of trypsin, EDTA After trypsin is complete.
Resus suspend the cells to a final volume of 10 milliliters. In supplemented dmm, remove any clumps from the cells by filtering them through a 100 micrometer cell strainer. After counting the cells resus, suspend them to the correct final concentration to begin the tube formation assay chill.
A 24 well plate and one milliliter tips for 20 to 30 minutes prior to loading. This prevents the BME from prematurely solidifying when ready to begin pipette 250 microliters of reduced growth factor BME into each well of a 24 well plate. Avoid creating bubbles by not depressing the pipette to its final stop.
When pipetting, once the wells are loaded, check to make sure that sufficient BME remains at the center of the well. When the meniscus forms, if the matrix is too thin, the cells will only form a monolayer. Next, incubate the plate for 30 minutes to solidify the BME.
Once the BME has set, mix approximately 300 microliters of conditioned media with 10 microliters of resuspended three B 11 cells and aliquot the cell suspension into the wells of the plate. After placing the plate in the incubator tubes will begin to form within two to four hours, Depending on the angiogenic factor concentration in the condition media peak tube formation may occur between three and 12 hours. The timing of the assay will need to be optimized for the individual study conditions.
Tubes will often begin to deteriorate within 18 hours, and the endothelial cells will undergo apoptosis At the time of peak tube formation. Carefully aspirate media from the well to avoid disrupting the tube network. Next, wash each well with one milliliter of DPBS for immediate visualization.
Add one milliliter of fresh DPBS to each well and image the tube network on a confocal microscope to fix a tube network for later visualization. Add 4%paraldehyde to each aspirated well and incubate for 15 minutes at room temperature After removing the para formaldehyde wash twice with DPBS and then add one milliliter of fresh DPBS to each. Well, these images show that angiogenic factors secreted by either mouse, keratinocytes or fibroblasts are capable of inducing tube formation.
Over time, endothelial cells migrate and begin to form small branches within one to two hours of plating. Maximum tube formation was reached by four to six hours. By 24 hours some branches remained, but many of the cells became apoptotic and tubes began to disconnect.
Cell number has a profound impact on tube formation. When an insufficient number of cells are seeded few tubes form and the network is minimal, as more cells are seeded, the tube network becomes more extensive. However, at too high of a concentration, the cells begin to clump or form monolayers and differences between treatment groups become masked, cells should have a minimum of two passages to ensure adequate tube formation.
The growth rate of three B 11 is so rapid that 25%of cells passage one day will yield a confluent flask the following day. Here, image J with the angiogenesis plugin is used on a calcine AM stain tube network to detect total nodes, tubes in mesh in combination, and merged on the network. Additionally, this program can be used to individually detect nodes, tubes, or mesh, which can be quantified.
After watching this video, you should have a good understanding of how to perform in vitro angiogenesis in a fast and reliable manner while avoiding common pitfalls.
