This method can answer key questions in the fields of hypertension and cardiovascular disease including the study of CD11c positive dendritic cells and how they potentiate the role of hypertensive injury. The main advantage of this technique is the isolation of CD11c positive dendritic cells from solid organs and the study of their physiological role in various disease states. So this process can be applied to multiple immune cell types, including B lymphocytes, T lymphocytes, monocytes, and macrophages.
Visualization of the technique is critical to understand dendritic cell isolation, specifically the adoptive transfer process into recipient mice. To begin, spray the chest and sides of the euthanized mouse with 70%ethanol. On the left side, carefully open the skin and the peritoneal cavity to expose the spleen.
Using small forceps, cautiously move the intestines and liver to the left side of the mouse. Then use small scissors to gently excise the spleen. Place each excised spleen into a labeled C tube containing three milliliters of RPMI 1640 medium.
First add collagenase D and DNase to prepared RPMI 1640 medium to prepare the spleen digestion solution. Place the C tube onto a semi-automated homogenizer and ensure that the tube is tightly placed on the rotating arm. Press the start button on the homogenizer to run the spleen 04.01 protocol for 60 seconds.
After this, detach the tube from the homogenizer and incubate the samples at 37 degrees Celsius under continuous at 20 rpm for 15 minutes. Next, use a pipette to transfer the solution into a 50 milliliter conical tube after filtering it through a 40 micrometer strainer. Wash the strainer with 10 milliliters of dPBS.
Centrifuge the cells at 300 times g and at four degrees Celsius for 10 minutes. Then aspirate the supernatant completely and wash the pellet by re-suspending it in 10 milliliter of dPBS. Centrifuge the suspension at 300 times g and at four degrees Celsius for 10 minutes.
Re-suspend the cell pellet in five milliliters of dPBS. Use a hemocytometer and Trypan blue exclusion to count the number of cells. Next centrifuge at 300 times g and at four degrees Celsius for 10 minutes to pellet the cells.
Aspirate the supernatant completely and re-suspend the pellet in 400 milliliters of Magnetically Activated Cell-sorting buffer. Then add 100 milliliters of CD11c microbeads per 100 million cells. Vortex the cell suspension and incubate for 10 minutes in a refrigerator at four degrees Celsius.
After this, add 10 milliliters of MACs buffer to wash the cells and centrifuge at 300 times g and at four degrees Celsius for 10 minutes. Aspirate the supernatant completely and re-suspend the cells in 500 milliliters of MACs buffer. Place the LS column in the magnetic field of the mag separator and place a 15 milliliter conical tube under each column to collect the flow through.
Next, rinse the columns with three milliliters of buffer. Place the cell suspension onto each column and collect the flow through which contains unlabeled cells. Wash the columns with three milliliters of MACs buffer and collect the unlabeled cells that pass through.
Wash the LS columns two additional times using three milliliters of MACs buffer per wash. After this, remove the LS columns from the magnetic separator. Add five milliliters of MACs buffer to each column and immediately plunge each column into a clear 15 milliliter conical tube to flush out the magnetically labeled cells.
Use a hemocytometer and Trypan blue exclusion to determine the CD11c positive DC number as outlined in the text protocol. Then dilute the cell sample at a one to one dilution in 0.4%Trypan blue solution. First, prepare the cell pellets in DC culture media as outlined in the text protocol.
Pipette 900 microliters of the DC suspension into 24-well flat bottom falcon plate. Add 100 microliters of the high salt DC culture media to each well, for a final sodium concentration of 190 millimolar. Label the plate and place it in a humidified carbon dioxide incubator at 37 degrees Celsius for 48 hours.
After 48 hours remove the plate from the incubator. Pipette the cell culture media in a single well up and down to re-suspend the CD11c positive DCs. Transfer all this suspension in a corresponding labeled 1.6 milliliter tube.
Repeat this for each individual well that was plated. Centrifuge all of the tubes at 300 times g and at four degrees Celsius for 10 minutes. Aspirate the supernatant and re-suspend the DC pellet with 100 microliters of sterile dPBS.
Next, draw the DC solution from the one of the tubes into a one milliliter syringe, equipped with a 27 gauge half-inch needle. Place the naive male 10 week old C57-black-6 mouse under 2%isoflurane to achieve a stable surgical plane. Check the level of anesthesia with a lack of pedal reflex.
Once a stable surgical plane is achieved, slowly and carefully introduce the needle into the retro-orbital space at an angle of approximately 30 degrees. Slowly and smoothly inject the CD11c positive DC suspension. Once the injection is complete, carefully remove the needle for the retro-orbital space, making sure not to damage the eye.
Then remove the mice from the nose cone and monitor them for approximately 30 minutes during recovery from anesthesia. A typical blood pressure analysis is shown here following the adoptive transfer of DCs and osmotic mini pumps that have been implanted for low dose Angiotensin II infusion. It should be noted that this low dose of Angiotensin II is a suppressor dose that does not increase blood pressure in a normal mouse.
Mice that receive normal salt-treated CD11c positive DCs maintain a normal blood pressure during the infusion, while the mice that receive high-salt treated CD11c positive DCs exhibit an increase in systolic blood pressure. Flow cytometry analysis is then performed utilizing the gating strategy shown here to determine the purity of the isolated CD11c positive cells. An increased enrichment of the CD11c positive cells is seen compared to the total splenocytes.
Different CD11c Microbead concentrations are utilized to troubleshoot the manufacturer's protocol. Magnetic separate of splenocytes using 100 microliters of Microbeads yields about 65%of CD11c positive cells, while using 200 microliters yields 55%and using 300 microliters yields 50%An FcR blocker is included along with 100 microliters of Microbeads. Following the magnetic separation, the blockade of FcR yields approximately 65%CD11c positive cells.
Some splenocytes are then incubated with 100 microliters of Microbeads and magnetically separated through an LS column. The separated cells are incubated with an additional 100 microliters of CD11c and separated again through an LS column. This double isolation process yielded 92%CD11c positive cells.
When attempting this procedure, it is important to confirm the purity of the isolated cells by flow cytometry. Following magnetic isolation of dendritic cells they can be cultured to determine their activation status by molecular techniques including proteomics and single-cell sequencing. Since its development, it is now possible to study the function and specific role of dendritic cells in hypertension.
Researchers are now exploring the specific antigens in hypertension.
