The al goal of the following experiment is to characterize the composition and phenotype of aortic and adventitional infiltrating leukocytes by flow cytometry. This is achieved by first isolating an atherosclerotic aorta as a second step. The surrounding adventitia is isolated from the aorta and single cell suspensions are prepared from both tissues.
Next, the single cell suspensions of fluorescently labeled to differentiate specific subsets of leukocytes from the advent tissue and aortic wall results are obtained that show the diversity of aortic infiltrating leukocytes during atherogenesis and the feasibility of isolating the aortic advent tissue leukocytes based on flow cytometric analysis of single cell suspensions. The main advantage of this technique over existing methods like immunohistochemistry is that leukocytes can be examined and phenotyped using multiple markers from a single aorta. This method can help answer key questions concerning the cellular mediators of inflammation in the pathogenesis of atherosclerosis and other vascular inflammatory disorders.
The identification of aortic leukocytes and phenotypical characterization of existing and new mediators of inflammation will help to ate the mechanisms of atherosclerosis. Place an empty collection tube for the blood and a collection tube containing PBS on ice. For each aorta to be collected, briefly soak a euthanized mouse with 70%ethanol and fasten the mouse to a dissection stage using laboratory tape.
Then using a heparinized syringe, draw blood from the mouse via cardiac puncture. Open the abdominal and chest cavities with a pair of dissecting scissors using a 10 milliliter syringe with a 25 gauge needle completely perfuse the vasculature with PBS containing 2%heparin. Next, dissect and remove the visceral organs, genital urinary organs, diaphragm and spleen, leaving the kidney's heart and aorta intact.
Make sure that there is no blood within the aortic tissues. Carefully dissect the adipose tissues and para aortic lymph nodes away from the aorta, leaving the aorta and adventitia intact. Then collect the whole aorta, including the aortic arch and descending, descending thoracic and abdominal portions.
Place the isolated aorta in a collection tube with PBS. Remove the PBS from the collection tube containing the aorta. Add 2.5 milliliters of one times aortic adventitia digestion enzyme solution to the aorta.
Then incubate the aorta with the enzyme solution for 10 to 20 minutes. At 37 degrees Celsius. Add PBS to a Petri dish during the incubation.
Following the incubation. Transfer the partially digested aorta from the enzyme solution to the Petri dish with the PBS very carefully. Using two pairs of curved forceps, peel the advent tissue layer away from the aorta as a single unit.
When the adventitia has been completely removed, transfer the adventitia and the aorta to separate fax tubes. Then add 2.5 milliliters of one times aorta dissociation enzyme solution to the aorta and adventitia. The aorta can be cut into smaller pieces.
To facilitate enzymatic digestion, incubate the aorta and adventitia for another 30 minutes at 37 degrees Celsius. Following this incubation, place the aorta and advent tissue tubes on ice. Prepare single cell suspensions from the aorta and adventitia using 70 micron cell strainers and syringe plungers to shear the remaining tissue apart.
Transfer the cell suspensions to five milliliter polypropylene Fax tubes. Pellet the cells by centrifugation for five minutes at 400 times gravity at four degrees Celsius. Reese has bend the cells in one milliliter of fax buffer and use trian blue and a hemo cytometer to count the cells.
Since the enzyme treatment may affect the expression of surface antigens. Incubate small pieces of spleen with or without the enzyme cocktail for one hour at 37 degrees Celsius after one hour. Confirm the lack of effect on the experimental surface antigen expression by flow cytometry.
Label an appropriate number of new experimental and control fax tubes. Add the experimental and control antibody cocktails for the desired extracellular antigens in a separate set of labeled tubes. Place the cocktail tubes on ice and cover them to protect the fluorophores from light.
Next, transfer an aliquot of five to eight times 10 to the five aortic and adventis cells into the corresponding pre-labeled fax tubes. Add one milliliter of fax buffer to each tube and pellet the cells by centrifugation. Remove the supan natant from the pelleted cells by decanting to discriminate between live and dead cells.
A live dead, fixable dead cell stain kit can be used first, create a one times live dead solution by adding one microliter of re dissolved live dead dye in one milliliter of PBS and vortex to mix. Next, add 100 to 200 microliters of one times live dead dye to the experimental and control sample tubes. Then incubate the samples with the diet room temperature for 10 minutes in the dark.
Incubate the single control tube for live dead at 56 degrees Celsius in the dark for 10 minutes to kill the cells by heat shock. Following the incubation, wash all the cells with one milliliter of PBS and pellet the cells by centrifugation. Next, wash the cells by adding one milliliter of fax buffer to each tube.
Vortexing the tubes to mix them and then pelleting the cells by centrifugation. Remove the supinate from the cells by decanting. Add 100 microliters of FC blockin.
Fax buffer to all of the tubes and gently vortex the tubes to resuspend the cells. Incubate the samples for 10 to 15 minutes at room temperature in the dark. Next, wash the cells as before.
Remove the SUP natum from the cells by decanting. Add 100 microliters of the appropriate antibody cocktail to its corresponding tubes. Gently vortex to resus.
Suspend the pellet and incubate all of the tubes for 20 to 30 minutes at room temperature in the dark following the 20 to 30 minute extracellular stain. Wash the cells twice as before, decant the supernatant and re suspend the pelleted cells in 300 microliters of 2%Para formaldehyde run the samples on a flow cytometer staining. The TER one 19 demonstrates that digested vessels not circulating peripheral blood are the source for most of the leukocytes analyzed in the aortic cell suspension.
TER 1 1 9 positive red blood cells account for 18%of the cells in this representative aortic cell suspension. This indicates that less than 0.02%of the cells isolated from aortic cell suspensions of blood derived the effects of the aorta dissociation enzyme cocktail on cyte surface antigens were assessed. The enzyme cocktail has no effect on the expression of CD 45 or CD 19 as seen here, as well as CD three TCR, alpha beta TCR Gamma delta, and several other surface antigens not shown in this figure.
In the next few figures, live aortic CD 45 positive leukocytes were gated to determine the percentage of interferon gamma positive T cells within the pool aorta of two young APO lipoprotein e deficient mice. Here, the forward and inside scatter profiles of a representative APOE aorta is shown to analyze leukocytes only the common leukocyte antigen CD 45 was used to gait on aortic leukocytes as seen here. The small granular population along the side scatter axis in the forward and side scatter profile of the aortic leukocytes seen in this figure suggests that necrotic cells are present in our leukocyte gait to remove these cells live dead aqua was used to gate on viable leukocytes, which made up to 30%of the representative population seen here in this figure.
The presence of small non granular cells in the CD 45 positive live dead negative forward and side scatter profile suggests that cellular fragments are still present in the CD 45 positive live dead negative gait. A polygonal forward side scatter gate was drawn to include only the larger viable cells in the analysis as seen here. Examination of the CD 45 positive live dead negative forward side scatter population for TCR Alpha Beta and interferon gamma reveals that 6%of the live aortic leukocytes are TCR alpha beta positive interferon gamma positive T helper one cells using a similar gating scheme as in the previous series of figures, representative intracellular staining data for CD 68 positive CD 11 B positive macrophages and interferon gamma positive TCR alpha beta positive cells from two A P OE null mice Fed Western diet for 12 weeks can be seen in the following figures as expected as seen in this figure.
The aorta of a Western diet fed APOE null mouse. The majority of leukocytes within the aorta are macrophages with 40%CD 68 and CD 11 B double positivity. Other CD 11 B positive myeloid cells represent 15%of the aortic leukocyte populates within the spleen of the same Western diet fed APOE null mouse.
7%of the viable SP cytes were CD 11 B positive CD 68 positive macrophages and 10%were other CD 11 B positive myeloid cells. In contrast in this figure of a representative, similarly stained isotype control sample only 0.01%of the viable APOE null cytes are falsely positive for the isotype. In addition, within 12 weeks in western diet fed APOE null mice, TCR alpha, beta positive interferon gamma negative T helper cells and interferon gamma producing TH one cells comprise a major proportion of the aortic infiltrating and splenic resident T-cell populations.
As seen in this figure examination of aov, no cyte stained with an isotype control suggests only 0.5%of TCR alpha beta positive interferon gamma positive cells are falsely positive for interferon gamma To demonstrate the feasibility of isolating aortic adventitia for flow cytometry staining here, representative CD 45 positive leukocyte staining for aortic and advent tissue cell suspensions are shown. Infiltrating CD 45 positive leukocytes represented approximately 18%of the adventitial cell suspension and 11%of the aortic cell suspension. While attempting this procedure, it's important to remember to keep those reagents and the cells on ice and in the dark unless otherwise noted.
After watching this video, you should have a good understanding of how to isolate and prepare single cell suspensions from the aorta and aortic adventitia or use in flow cytometry experiments.
