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. 
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<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...

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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 This method is not restricted to murine aortas and we (unpublished data) and others10 used this approach to analyze human specimens such as internal mammary artery, aortic valves and coronary arteries.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...

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		<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 ...

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