The overall goal of this experiment is to obtain large numbers of highly purified high quality mouse coronary endothelial cells. This method can help answer key questions in the field of cardiovascular research. The main advantage of this technique is that it facilitates the acquisition of a high yield of good quality endothelial cells.
Demonstrating this procedure will be Shizhen Luo, a research specialist, and Dr.Ayako Makino, our principal investigator. Up to one week before the procedure, add one milliliter of CD31 buffer to one 1.5 milliliter microcentrifuge tube per sample. Next, mix sheep antirad IGG beads with gentle pipetting, and add the appropriate volume of beads to each tube.
After all of the beads have been added, shake the tubes vigorously 30 times and place them on a magnetic plate for one minute of shaking. Then, with the tubes still attached to the plate, discard the buffer and add one milliliter of fresh CD31 buffer to each tube. Add two microliters of rad anti-mouse CD31 antibody and place the tubes on a rotator at four degrees Celsius overnight.
The next day, transfer the tubes under a laminar flow hood and place them back onto the magnetic plate with shaking for one minute. Then replace the supernatant with one milliliter of fresh CD31 buffer and shake the tubes vigorously by hand 30 times. After the third wash, return the tubes to the rotator at four degrees Celsius.
On the day of the procedure, begin by injecting the experimental animal intraperitoneally with 0.1 milliliters of heparin to prevent blood coagulation. After ten minutes, anesthetize the mouse with an injection of 0.01 milliliters pentobarbital. Then, confirm a lack of response to toe pinch.
After removing the heart and lung tissue together, place them into a beaker of Krebs buffer and rinse the tissues with gentle shaking. Then transfer the tissues into a six centimeter dish containing fresh Krebs, and use scissors and foreceps to remove the lung lobes. Now insert a 20 gauge catheter into the aorta and flush the coronary circulatory system with HBSS supplemented with heparin.
When all of the blood has been removed, make a small cut in the ventricle, rinse the heart with gentle shaking, and place the cardiac tissue into a sample tube on ice. After digesting the tissue to release the coronary endothelial cells, pellet and wash the cells two times with five milliliters of washing buffer. Then place a tube of the previously prepared beads onto the magnetic plate and shake the tube three times to evenly distribute the beads.
After the second wash, remove as much supernatant as possible without disrupting the pellet, and associate the cells with vigorous flicking. Next, resuspend the cells in one milliliter of washing buffer with gentle mixing and discard the CD31 buffer from the beads. Combine the cells with the beads, and flick the tube 30 times.
Then place the cell and bead solution onto the rotator at four degrees Celsius for 30 minutes. While the cells are incubating, coat the surface of one plating chamber well per sample with gelatin solution, and place the chambers at 37 degrees Celsius for 30 minutes. When the gelatin has solidified, remove any excess gel solution and dry the chamber well surface.
Then transfer the bead conjugated cells to the magnetic plate under the hood for two minutes of shaking, and discard the washing buffer. Add one milliliter of fresh washing buffer to the cells and remove the tube from the plate for 30 vigorous shakes, followed by 30 flicks. Return the tube to the plate for another minute of shaking.
After the last agitation, return the tube to the magnetic plate for a final minute of shaking, and replace the washing buffer with one milliliter of 37 degree Celsius endothelial cell medium, supplemented with 20 percent iron fortified calf serum. Shake and flick the tubes 30 final times. Then use a one milliliter pipetter set to 500 microliters to agitate the conjugated cells ten times, and transfer 500 microliter aliquots of coronary endothelial cells to each plating chamber well for overnight incubation at 37 degrees Celsius.
The next day, wash the cells in endothelial cell medium, supplemented with ten percent FBS. The purity of the coronary endothelial cells isolated by this method can be confirmed by immunohistochemical staining against the endothelial cell surface markers, BS lectin, and AC LDL, with over 90 percent of the cells demonstrating a double positive expression of both markers in a typical mouse coronary endothelial cell culture. Following this procedure, as a method, various blood, immunoreception, and fluorescent imaging can be performed to answer additional questions, such as what protein can be modified during different disease states.
After its development, this technique provides the tools necessary for researchers to expand their research into the field of coronary endothelial cells. After watching this video, you should have a good understanding of how to isolate mouse coronary endothelial cells of high quantity and quality. There are several key points during this procedure.
The most important thing is to maintain a sterile environment throughout the isolation. Second is to flush the blood completely and as soon as possible to avoid blood coagulation in the capillary network. Third is to measure the pH of all the buffer, especially CD31 buffer.
