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

这项工作是由国家批卫生研究院：PO1 HL55798（吉隆坡）和美国心脏协会的科学家开发授予0525532U（EG）的支持。

1。分离小鼠主动脉机构IACUC委员会批准的程序是必需的工作与小鼠。 <ol><li>准备加入肝素钠1000个单位，50毫升PBS和反相混合管heparinzed PBS。准备一个空的收集管的血液和集合管中，PBS为每个要收集的主动脉。所有管置于冰上。 </li><li> Heparinize一个注射器吸取血（1000 U / ml的肝素钠0.1毫升），准备手术工具（两对弯钳，一双解剖剪刀，和一双的microshears），并为解剖解剖阶段。...

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	<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>

在这里，我们提出了一个基于流式细胞仪检测的免疫细胞组成的小鼠主动脉的调查方法。这种方法的主要优点是能够分析主动脉免疫细胞在单细胞水平和表征主动脉白细胞的激活状态。这种方法不仅限于小鼠主动脉和我们（未发表数据）和其他 10用这种方法来分析人体标本，如乳内动脉，主动脉瓣和冠状动脉。这种方法的一个限制是恢复单一主动脉小鼠主动脉白细胞数量相对较少。虽然?...

<table border="1"><tbody><tr><td> 材料 </td><td> 产品编号 </td><td> 公司 </td></tr><tr><td>胶原酶第十一</td><td> C7657 </td><td> Sigma - Aldrich公司</td></tr><tr><td>透明质酸</td><td> H3506 </td><td> Sigma - Aldrich公司</td></tr><tr><td>我脱氧核糖核酸酶，2型</td><td> D4527 </td><td> Sigma - Aldrich公司</td></tr><tr><td>胶原酶我</td><td> C0130 </td><td> Sigma - Aldrich公司</td></tr><tr><td>胶原酶二</td><td> LS004174 </td><td>沃辛顿生化公司</td></tr><tr><td>弹性蛋白酶</td><td> LS002292 </td><td>沃辛顿生化公司</td></tr></tbody></table>

flow cytometry analysis of immune cells within murine aortas

动脉粥样硬化是一种慢性炎症过程大中型船只的特点是形成泡沫细胞，免疫细胞，血管内皮细胞和平滑肌细胞，血小板，细胞外基质，具有广泛坏死富含脂质核心组成的斑块和周围组织纤维化1先天免疫和适应性免疫反应的武器是参与动脉粥样硬化的启动，发展和持久2，3是一个显着的证据机构，不同亚群的免疫细胞，如巨噬细胞，树突状细胞，T和B淋巴细胞，在目前的健康，易发生动脉粥样硬化小鼠 4主动脉。此外，免疫细胞被发现在周围，这表明这个组织的重要作用，在动脉粥样硬化的主动脉外膜2。 一段时间以来，不同类型的免疫细胞，它们的激活状态，和主动脉壁内细胞成分的定量检测RT - PCR和免疫组织化学方法对动脉粥样硬化的研究是有限的。作了一些尝试执行流式细胞仪利用人体的主动脉，和一些问题，如高自体荧光，已报告 5,6 。人类动脉粥样硬化斑块1胶原酶消化，收集游离细胞和CD14的染色+ /表面CD11c +突出的巨噬细胞源性泡沫细胞。在这项研究中，一个“模拟”通道使用，以避免假阳性染色。6坏死性材料，在消化过程中积累了大量的碎片，产生在主动脉样品的自体荧光上升。要解决这个问题，阴性和阳性对照小组已经提出，但只有双染色可以应用于这些样本。我们已经开发出一个新的流式细胞仪为基础的方法，分析免疫细胞的组成和特征的活化，增殖，免疫细胞分化的健康，易发生动脉粥样硬化主动脉。此方法使主动脉壁的免疫细胞组成的调查，并打开使用广谱的免疫学方法对这种疾病的免疫方面的调查的可能性。

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

本文介绍一个基于流式细胞仪的方法进行调查的主动脉免疫组成。该文件还说明了一个额外的技术，允许单独审视周围的外膜和血管壁。这种方法打开执行主动脉白细胞表型分析，适用于动脉粥样硬化研究的几种免疫检测的可能性。

流式细胞仪分析小鼠主动脉内流的免疫细胞

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

在固定和活细胞，流式细胞仪测量钙蛋白酶活性

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

科研

免疫与感染

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

人类<em在体外承认免疫失衡的早期国家禁止作为筛检工具

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

主要鼠Ⅱ型肺泡上皮细胞的功能及分子生物学研究的流式细胞仪分离

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

细胞内染色和流式细胞仪识别鼠主动脉，肾脏和淋巴结内淋巴细胞亚群的高血压模型

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

影像性流式细胞仪对人体免疫突触的定性和定量分析

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

小鼠与人肺泡巨噬细胞的分离和体外培养

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

双氟铬流式细胞仪对七种免疫细胞子集的判别

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

全骨髓的制备,用于中性粒系细胞的大规模细胞测定分析

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

小鼠肾-居民CD8+T 细胞的分离，用于流细胞测量分析

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

流式细胞术分析人髓系U937细胞中SAMHD1限制性

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