The overall goal of this in-vivo matrix gel assay is to investigate the interaction of endothelial cells with parasites. This matrigel assay is designed to help investigators study the major steps in androgenesis. It also will allow the identification of signaling pathways responsible for major steps in blood vessel formation as well as genes who's disfunction can lead to vascular disorders and other aberrations of androgenesis.
The main advantage of this technique is that it allows us to study the in-vivo blood vessel formation involving both endothelial cells and their support cells, pericytes. To begin, grow human endothelial and parasite cell cultures on 100-millimeter plates with 10 milliliters of the appropriate media. Change the media every two to three days until the cells reach 80%confluency.
In order to prepare the matrix gel solution to mix with the cells, thaw a stock of matrix gel overnight at four degrees Celsius. Then make 1.5 milliliter aliquots for storage at negative-20 degrees Celsius long-term, or at four degrees Celsius for up to two weeks. Each experimental plug will require 200 microliters of matrix gel.
So one stocked tube can supply six plugs with a 25%excess. Prepare each required aliquot for use by adding 15 microliters of thawed 100x beta-FGF stock. Mix the beta-FGF into solution by pipetting, then incubate on ice for at least 30 minutes prior to adding cells.
At this point, chill some 28-gauge 1cc insulin syringes for the injections. To collect the cells from the culture plates, remove the media and wash the cells once with PBS. After removing the PBS, add one milliliter of trypsin solution and return the plates to the incubator for one minute.
Then, pipette two milliliters of culture media over the plates to detach the cells and collect the medium into a 15 milliliter tube. Now, check the concentration of the collections using a hemocytometer. Then, prepare aliquots of cells in 15 milliliter tubes.
A combination of one million endothelial cells and 200, 000 parasites is used here for the experimental group. Next, centrifuge the cell aliquots at 400g for five minutes. Then, add the calculated volume of matrix gel to the cell pellet, and mix with gentle pipetting so no foam is made, as air bubbles cannot be injected.
Now keep the preparations on ice until the mice are ready to inject. After anesthetizing a mouse with isoflurane, and confirming the anesthetized state with a toe pinch, attach the mouse to the non-rebreathing system supplying isoflurane and place it on the heating pad, ventral side down. Now remove about a square-centimeter of hair from both lateral hind-regions.
Then, wipe the exposed skin clean using 70%alcohol. Next, load one milliliter of the prepared cell solution into a chilled syringe, which is enough for four plugs. To make an injection, lift the back-skin to locate the subcutaneous space.
Then slowly inject 200 microliters evenly into the subcutaneous space posterior of the ribcage, and slowly remove the needle to prevent leaking. After the injection, a bump will form at the site. Wipe the site with alcohol, then outline the bump with permanent marker so it can be found later.
Then, repeat the injection procedure on the other side of the mouse. Be sure to inject all the cells within three minutes or they will start sticking to the syringe. The injection step is critical to do well to ensure that the matrix gel plug will form between the skin and muscle layers so that the plug maintains good shape and it does not become diffuse.
Before proceeding to inject the next mouse, let the injected mouse stay on the heat pad for a minute to give the matrix time to gel. After euthanasia, set-up the mouse on the operating stage. After removing any regrown hair from the injection site, find the marker lines that identify the location of the plug.
Then, using fine surgical scissors, make an incision along the marked border of the plug, perpendicular to the skin and through to the muscle beneath. Carefully cut around the periphery of the marked border. Then remove the plug, keeping it sandwiched between the skin and muscle layers.
Immediately rinse the plug off in a small beaker of PBS to remove the blood. Then, place the entire plug in a 50 milliliter tube with 25 milliliters of 4%paraformaldehyde. Let it fix at room temperature for 24 hours without any agitation.
The next day, transfer the plugs into 25 milliliters of 70%ethanol for 24 hours. Then, proceed with paraffin embedding. Sections of plugs containing only endothelial cells show that some vessels do not get perfused with the host's blood after 14 days in-vivo.
By contrast, plugs containing both endothelial cells and parasites, display many perfused vessels after 14 days in-vivo. These plugs have cells that are positive for smooth muscle actin staining, seen in red, immediately adjacent to CD31 positive endothelial cells, seen in green. Therefore, parasites may play a substantial role in nascent blood vessel formation.
After watching this video, you should have a good understanding of how to apply this powerful in-vivo technique to study blood vessel formation in use in multiple cell types. This technique will pave the way for researchers to explore various topics of vascular biology in mouse models. Following this procedure, other methods, such as quantitative PCR, and Western immunoblotting can also be run to answer additional questions such as changes in gene expression and proteomics.
This powerful technique will allow the investigator to study in-vivo how endothelial cells and parasites interact during blood vessel formation. Which is a key step in androgenesis. This powerful technique will also allow the investigator to study other cell-cell interactions that are relevant to understanding vascular diseases such as pulmonary hyper-tension, among others.
