Name: flow cytometry analysis of immune cells within murine aortas
Uploaded: 2011-07-01T13:00:00
Description: Watch this Scientific Journal Video about Flow Cytometry Analysis of Immune Cells Within Murine Aortas at JoVE.com

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.).

1. Isolation of murine aortas 
Institutional IACUC committee approval of the procedure is required to work with mice. 
<ol>
<li>Prepare heparinzed PBS by adding 1000 units of heparin sodium to 50 ml PBS and inverting the tube to mix. Prepare an empty collection tube for the blood and a collection tube containing PBS for each aorta to be collected. Keep all tubes on ice.</li>
<li>Heparinize a syringe for drawing blood (0.1ml of 1000 U/ml Heparin sodium), prepare surgical tools (two pairs of c...

<ol>
	<li>Lusis, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atherosclerosis.">Atherosclerosis.</a> Nature. 407, 233-2341 (2000).</li><li>Galkina, E., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+and+inflammatory+mechanisms+of+atherosclerosis.">Immune and inflammatory mechanisms of atherosclerosis.</a> Annu Rev Immunol. 27, 165-197 (2009).</li><li>Hansson, G. K., Libby, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immune+response+in+atherosclerosis:+a+double-edged+sword.">The immune response in atherosclerosis: a double-edged sword.</a> Nat Rev Immunol. 6, 508-519 (2006).</li><li>Galkina, E., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+influx+in+atherosclerosis.">Leukocyte influx in atherosclerosis.</a> Curr Drug Targets. 812, 1239-1248 (2007).</li><li>Bonanno, E., Mauriello, A., Partenzi, A., Anemona, L., Spagnoli, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+analysis+of+atherosclerotic+plaque+cells+from+human+carotids:+a+validation+study.">Flow cytometry analysis of atherosclerotic plaque cells from human carotids: a validation study.</a> Cytometry. 39, 158-165 (2000).</li><li>Liu-Wu, Y., Svenningsson, A., Stemme, S., Holm, J., Wiklund, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+analysis+of+macrophage-derived+foam+cells+from+human+atherosclerotic+lesions+by+using+a+"mock"+FL3+channel+in+flow+cytometry.">Identification and analysis of macrophage-derived foam cells from human atherosclerotic lesions by using a "mock" FL3 channel in flow cytometry.</a> Cytometry. 29, 155-164 (1997).</li><li>Galkina, E., Kadl, A., Sanders, J., Varughese, D., Sarembock, I. J., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte+recruitment+into+the+aortic+wall+before+and+during+development+of+atherosclerosis+is+partially+L-selectin+dependent.">Lymphocyte recruitment into the aortic wall before and during development of atherosclerosis is partially L-selectin dependent.</a> J Exp Med. 203, 1273-1282 (2006).</li><li>Tung, J. W., Parks, D. R., Moore, W. A., Herzenberg, L. A., Herzenberg, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+approaches+to+fluorescence+compensation+and+visualization+of+FACS+data.">New approaches to fluorescence compensation and visualization of FACS data.</a> Clin Immunol. 110, 277-283 (2004).</li><li>Smith, E., Prasad, K. M., Butcher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blockade+of+interleukin-17A+results+in+reduced+atherosclerosis+in+apolipoprotein+E-deficient+mice.">Blockade of interleukin-17A results in reduced atherosclerosis in apolipoprotein E-deficient mice.</a> Circulation. 121, 1746-1755 (2010).</li><li>Eid, R. E., Rao, D. A., Zhou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-17+and+interferon-gamma+are+produced+concomitantly+by+human+coronary+artery-infiltrating+T+cells+and+act+synergistically+on+vascular+smooth+muscle+cells.">Interleukin-17 and interferon-gamma are produced concomitantly by human coronary artery-infiltrating T cells and act synergistically on vascular smooth muscle cells.</a> Circulation. 119, 1424-1432 (2009).</li></ol>

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 met...

<table border="1">
	<tbody>
		<tr>
			<td>Material</td>
			<td>Catalog Number</td>
			<td>Company</td>
		</tr>
		<tr>
			<td>Collagenase XI</td>
			<td>C7657</td>
			<td>Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>H3506</td>
			<td>Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>DNase I, type 2</td>
			<td>D4527</td>
			<td>Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>Collagenase I</td>
			<td>C0130</td>
			<td>Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>Collagenase II</td>
			<td>LS004174</td>
			<td>Worthington Biochemical Corporation</td>
		</tr>
		<tr>
			<td>Elastase</td>
			<td>LS002292</td>
			<td>Worthington Biochemical Corporation</td>
		</tr>
	</tbody>
</table>

flow cytometry analysis of immune cells within murine aortas

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.

Watch this Scientific Journal Video about Flow Cytometry Analysis of Immune Cells Within Murine Aortas at JoVE.com

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Research

JoVE Journal

Immunology and Infection

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Intracellular Staining and Flow Cytometry to Identify Lymphocyte Subsets within Murine Aorta, Kidney and Lymph Nodes in a Model of Hypertension

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the ...

Analysis of SAMHD1 Restriction by Flow Cytometry in Human Myeloid U937 Cells

Sterile &#945;-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are ...