1Deptartment of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, 2Division of Inflammation Biology, LaJolla Institute for Allergy and Immunology
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Butcher, M. J., Herre, M., Ley, K., Galkina, E. Flow Cytometry Analysis of Immune Cells Within Murine Aortas. J. Vis. Exp. (53), e2848, doi:10.3791/2848 (2011).
Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.
1. Isolation of murine aortas
Institutional IACUC committee approval of the procedure is required to work with mice.
2. Preparation of single cell suspensions
3. Flow cytometry staining
4. Flow cytometry analysis of isolated surrounding adventitia and vessel wall
As leukocytes can migrate to the aortic adventitia as well as atherosclerotic plaques within the aortic wall, to examine adventitial and aortic leukocytes by flow cytometry a protocol for isolating and performing flow cytometry on these two anatomical sites was developed.9 Briefly, before the whole aortas are digested with ADES (Section 2, step 2) the aortic adventitia is partially digested and removed from the rest of the vessel. Once the adventitia is removed and set aside, the rest of the aorta is digested with ADES to liberate leukocytes from the vessel wall.
5. Representative Results
Here we present a number of figures that demonstrates flow cytometry staining to analyze the immune composition of whole aortas, the aortic vessel wall and the surrounding aortic adventitia. First, we demonstrate a representative FACS plot that shows a TER-119 staining on the whole blood and isolated aortic cell suspension (Fig. 1). TER-119 positive red blood cells accounted for 18% of the cells in the aortic cell suspension, which indicates that only 0.014% of the cells isolated from aortic cell suspensions are blood-derived. This is an important control experiment that clearly demonstrates that digested vessels, but not circulating peripheral blood is the source for most leukocytes analyzed in the aortic cell suspension. In addition, to validate the method, we assessed the effects of the aorta dissociation enzyme cocktail on splenocyte surface antigens (Fig. 2). The enzyme cocktail has no effects on the expression of several antigens, including, CD45, CD19, CD3, TCRαβ, TCRγδ and several other surface antigens7.
CD45 staining in conjunction with Live/Dead viability dye and appropriate isotype controls are used to detect and sub-gate major populations of interest and to exclude cellular debris during analysis. In the Fig.3, live aortic CD45+ leukocytes were gated to determine the percentage of IFNΥ+ T cells within the pooled aortas of two young Apoe-/- mice. To emphasize the versatility of this method, using the gating scheme presented in Fig.3, we present in Fig.4 representative intracellular staining for CD68+CD11b+ macrophages and IFNγ+TCRαβ+ cells from two Apoe-/- mice fed western diet for 12 weeks. As expected, in western diet fed Apoe-/- aortas, the majority of the leukocytes within the aorta are macrophages (40% CD68+CD11b+) or other myeloid cells (CD11b+CD68low and CD11b+CD68-). In addition, Th1 cells comprise a major portion of the aortic infiltrating TCRαβ T cells (Fig.4). As the overall percentage of aortic leukocytes and leukocyte subsets varies depending on the age of the mouse and severity of atherosclerosis, the optimal gating strategy for major populations of interest should be empirically determined
To demonstrate the feasibility of isolating aortic adventitia for flow cytometry staining, we present representative CD45+ leukocyte staining for aortic and adventitial cell suspensions (Fig 5). Briefly, three aged Apoe-/- mouse aortas were digested and pooled together as described above (Sections 2 and 4). Infiltrating CD45+ leukocytes were detected in both the adventitia (18% of the cell suspension) and the remaining aorta (11% of the cell suspension).
Figure 1.TER-119 staining in digested aortic cell suspension. Aorta was perfused by cardiac puncture with PBS containing 2% heparin. Then the aorta was digested with the enzyme cocktail for 1 hr at 37°C. Aortic cells suspension and blood sample (as a positive control) were stained with anti-TER-119-PE Abs and analyzed by flow cytometry. TER-119-positive red blood cells account for 18% of all cells in the aortic cell suspension indicating that only 0.014% of all leukocytes isolated from aortas are likely to be blood-derived leukocytes.
Figure 2.Enzyme-cocktail treatment has no effects on CD45 (top) or CD19 (bottom) expression on lymphocytes. Cell suspensions from untreated (A, B) and treated with enzyme-cocktail (C, D) LN were obtained, and stained with APC-Cy7-conjugated anti-CD45 and APC-conjugated anti-CD19 mAbs. (A, C) The numbers represent the percentages of CD45+ leukocytes in R1 gate. (B, D) The numbers represent the percentages of CD45+/CD19+ lymphocytes. Profiles are gated on CD45+ leukocytes.
Figure 3. Gating strategy for the analysis of aortic leukocytes. Two aortas were isolated and pooled from atherosclerotic prone Apoe-/- mice and aortic cell suspensions were prepared as described above. The cell suspensions were stained for CD45 (PerCP), TCRαβ (FITC), IFNγ (eFluor 450), and Live/Dead Aqua, and analyzed using a Cytek DXP 8 Color upgraded BD FACS Calibur. Briefly, CD45+ Leukocytes were gated (B) and further analyzed (C-F). Dead cells were removed from the analysis based on Live/Dead Aqua staining (D) and FSC plots (E). Live aortic leukocytes were then examined for TCRαβ and IFNγ expression (F).
Figure 4. Intracellular staining for intracellular antigens and cytokines. Cell suspensions were prepared from a whole Apoe-/- aorta and spleen as described. Aortic (A) and splenic (B, C) cell suspensions were stained for CD45, CD11b, and CD68 or an isotype control using BD Cytofix/Cytoperm™ Kit (BD Biosciences). Cells were gated on CD45+ leukocytes and debris was excluded based on the forward and side scatter profiles. For intracellular IFNγ staining, aortic and splenic cell suspensions were cultured for five hours in RPMI 1640 supplemented with Golgi stop, PMA, and Ionomycin C, as earlier described.9 Stimulated single aortic (D) and splenic (E, F) cell suspensions were subsequently stained with CD45(PerCP), TCRαβ (FITC), CD3 (APC-Cy7), Live Dead Aqua, and IFNγ (eFluor 450; E) or an isotype (IgG1-eF450, F). T cells (D-F) were gated from live CD45+CD3+ leukocytes (CD45+Live/Dead Aqua-) and examined for TCRαβ and IFNγ.
Figure 5. Representative image of isolated murine aortic adventitia and aortic vessel wall. Representative flow cytometry counter plot demonstrates the presence of CD45+ T cells in the adventitia and aortic wall of aged Apoe-/- mice. SSC- side scatter. To eliminate autofluorescence from debris and necrotic tissues, plots were gated for FSC>750. To avoid additional autofluorescence from doublets the gates were also set up as FSC<3500, SSC<3500. The percentages indicate CD45+ cells in the gates.
Here, we present a flow-cytometry-based method for the investigation of the immune cell composition of murine aortas. The major advantage of this method is the ability to analyze aortic immune cells at a single cell levels and to characterize the activation status of aortic leukocytes. This method is not restricted to murine aortas and we (unpublished data) and others10 used this approach to analyze human specimens such as internal mammary artery, aortic valves and coronary arteries. One limitation of this method is the relatively low number of murine aortic leukocytes recovered single aortas. While the number of leukocytes recovered from a single atherosclerotic aorta is sufficient for a flow cytometry experiment, cells which are low in abundance may be difficult to detect in a single aortic cell suspension. If necessary, this problem can be circumvented by using combined samples from 2-4 aortas for flow cytometry staining. In addition, since the enzyme treatment may affect the expression of surface antigens, the resistance to enzyme treatment should be determined for all antigens of interest. We have previously validated several antigen markers, which are unaffected by the enzymatic digestion7. However, untested antibody clones should be validated by the investigator before being used in flow cytometry experiments7.
Competitive adoptive transfer homing assay is a powerful method to analyze the kinetics and mechanisms of leukocyte recruitment to a site of inflammation. We successfully applied flow cytometry analysis of aortas from recipient mice to investigate mechanism of leukocyte migration to the aortas.7 To analyze recently proliferated cells, the incorporation of bromodeoxyuridine (Brdu) in vivo can be used as an excellent marker for proliferating cells. We applied this technique with following flow cytometry analysis to detect antigen-specific T cells in the aortas of the recipient mice.7
There is a growing body of evidence suggesting that the aortic adventitia plays an important role in atherogenesis. Immunohistochemical staining of aortas with the surrounding adventitia provides essential data about leukocyte localization within the analyzed tissues, but has some limits in the detailed characterization of leukocyte subpopulations. We developed a flow cytometry based method that permits the analysis of the aorta and surrounding adventitia separately9 This exciting new flow cytometry-based technique, in conjunction with well-established histology and molecular-based techniques, will help to identify possible differences in phenotypical and functional characteristics of adventitial and vessel wall residing leukocytes.
No conflicts of interest declared.
This work was supported by National Institutes of Health grant: PO1 HL55798 (to K.L.) and American Heart Association Scientist Development Grant 0525532U (to E.G.).
|DNase I, type 2||Sigma-Aldrich||D4527|
|Collagenase II||Worthington Biochemical||LS004174|