Name: intracellular staining and flow cytometry to identify lymphocyte subsets within murine aorta, kidney and lymph nodes in a model of hypertension
Uploaded: 2017-01-28T13:00:00
Description: Watch this Scientific Journal Video about Intracellular Staining and Flow Cytometry to Identify Lymphocyte Subsets within Murine Aorta, Kidney and Lymph Nodes in a Model of Hypertension at JoVE.com

这项工作是由美国心脏协会奖学金奖（16POST29950007）到FL，从健康（NIH T32 HL069765），以BLD，美国心脏协会奖学金奖（14POST20420025）至MA萨利赫全国学院培训津贴，以及美国国立卫生研究院支持K08奖（HL121671），男男性接触者。 MSM也由吉利德科学公司研究基金支持

范德比尔特大学的机构动物护理和使用委员会已批准本文所述的程序。小鼠被收容并按照指南实验动物的护理和使用照顾（美国国家科学院出版社2010年修订）。 1.主动脉淋巴结，肾和主动脉的小鼠隔离<ol><li>安乐死的CO 2吸入老鼠。喷用70％乙醇的胸部和小心地打开皮肤和胸壁用剪刀以暴露心脏。 </li><li>灌注脉管，在右心房执行一个小切口，并稳定地注入至少10毫升冷的PBS（约1毫升...

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation,+Immunity,+and+Hypertensive+End-Organ+Damage.">Inflammation, Immunity, and Hypertensive End-Organ Damage.</a> Circ Res. (6), 1022-1033 (2015).</li><li>Luckheeram, R. V., Zhou, R., Verma, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4+T+Cells:+Differentiation+and+Functions.">CD4+T Cells: Differentiation and Functions.</a> Clin Dev Immunol. 2012, 1-12 (2012).</li><li>Mittr&#252;cker, H. -. W., Visekruna, A., Huber, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+in+the+differentiation+and+function+of+CD8++T+cells.">Heterogeneity in the differentiation and function of CD8+ T cells.</a> Arch Immunol Ther Exp (Warsz). 62 (6), 449-458 (2014).</li><li>Autengruber, A., Gereke, M., Hansen, G., Hennig, C., Bruder, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+enzymatic+tissue+disintegration+on+the+level+of+surface+molecule+expression+and+immune+cell+function.">Impact of enzymatic tissue disintegration on the level of surface molecule expression and immune cell function.</a> Eur J Microbiol Immunol (Bp). 2 (2), 112-120 (2012).</li><li>Finak, G., Langweiler, M., Jaimes, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardizing+Flow+Cytometry+Immunophenotyping+Analysis+from+the+Human+ImmunoPhenotyping+Consortium.">Standardizing Flow Cytometry Immunophenotyping Analysis from the Human ImmunoPhenotyping Consortium.</a> Sci Rep. 6, 20686 (2016).</li><li>Herzenberg, L. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+genomics.">Single-cell genomics.</a> Nat Methods. 8 (4), 311-314 (2011).</li><li>Chattopadhyay, P. 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T., Yosef, N., Lee, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Cell+Genomics+Unveils+Critical+Regulators+of+Th17+Cell+Pathogenicity.">Single-Cell Genomics Unveils Critical Regulators of Th17 Cell Pathogenicity.</a> Cell. 163 (6), 1400-1412 (2015).</li></ol>

The protocol described herein has been optimized to properly identify T cell subsets present within murine kidneys, aorta and lymph nodes. This protocol can be easily adapted to examine other immune cell subsets such as B lymphocytes and innate immune cells and can be modified to include other tissue types. The digestion step is critical and has to be modified and optimized for each tissue9. A prolonged digestion step or the use of an inappropriate enzyme can affect the stability of antigen expression. Similar...

最近的证据表明，适应性免疫系统，特别是T淋巴细胞，在几个心血管疾病1,2,3,4,5的发展中发挥了关键作用。例如，在血管紧张肽II引起的高血压模型中，T细胞在血管和小鼠的肾脏的累积已经描述6。血管积累主要是在外膜和血管周围的脂肪。在肾脏中，T细胞堆积在髓质和肾皮质两者。取决于哪个子集参与，这些T细胞产生不同的细胞因子，可以影响血管和肾功能，并导致病理的发展（由麦克马斯特等 6中综述）。 CD4 + T辅助淋巴细胞可分为几个子集：根据其职能和签名CYTO T辅助1（Th1细胞），Th2细胞，TH9，Th17细胞，TH22，调节性T（Treg细胞）细胞和T滤泡辅助（Tfh发挥细胞）基恩斯7。同样，CD8 +细胞毒性T细胞可以被分类为TC1，TC2，TC17或TC9 8。也有（不表达的CD4或CD8 T细胞的标志物即细胞）双阴性T细胞。这些细胞的一个子集具有一个备用的γδT细胞受体（而不是经典α和β受体），因此，被称为的γδT细胞。通过表面标记，细胞因子和转录因子的流式细胞仪的多参数分析构成，以确定这些细胞中的最好的方法。虽然这种方法是在免疫学领域广泛使用，这是不那么在实体器官和在心血管疾病的设置说明。 从历史上看，在组织中的淋巴细胞的识别仅限于免疫组化或RT-PCR的方法。虽然免疫组织化学和免疫功能强大的方法来确定一个int抗原的组织分布erest，他们是不足以表型鉴定所涉及的子集。此外，虽然RT-PCR分析是检测抗原，细胞因子或转录因子的mRNA表达是有用的，它不允许在单个细胞水平同时多种蛋白质的检测。 流的出现术，特别是当与细胞内染色组合以检测细胞因子和转录因子，提供了强大的技术，其允许鉴定和定量在固体器官的免疫细胞亚群的单个细胞水平的调查。我们已经优化了细胞内染色法，流式细胞仪鼠肾，主动脉和主动脉淋巴结内出现在血管紧张素II诱发的高血压模型中的主要T细胞亚群鉴别。在一个高度重新组织消化， 离体活化，透化，并且表面和细胞内染色结果：每一步的优化可生产测定可应用于其他心血管和肾脏疾病模型。

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			<td height="21" style="height:21px;width:381px;">Collagenase D</td>
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			<td height="21" style="height:21px;">Collagenase A</td>
			<td>ROCHE</td>
			<td>10103586001</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Collagenase B</td>
			<td>ROCHE</td>
			<td>11088815001</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Dnase</td>
			<td>ROCHE</td>
			<td>10104159001</td>
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		<tr height="21">
			<td height="21" style="height:21px;">1X Red blood cell lysis buffer</td>
			<td>eBioscience</td>
			<td>00-4333-57</td>
			<td></td>
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			<td height="21" style="height:21px;">RPMI Medium 1614 1X</td>
			<td>Gibco</td>
			<td>11835-030</td>
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			<td height="21" style="height:21px;">Percoll</td>
			<td>GE Healthcare</td>
			<td>17-5445-02</td>
			<td>For density gradient centrifugation</td>
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			<td height="21" style="height:21px;">GentleMACS &#8482; C tube&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-334</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GentleMACS dissociator device</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-235</td>
			<td>Use the program SPLEEN_04</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Cell activation cocktail (with Brefeldin A)</td>
			<td>Biolegend</td>
			<td>423303</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">anti-CD16/32</td>
			<td>eBioscience</td>
			<td>14-0161-81</td>
			<td>dilute 1:100</td>
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		<tr height="21">
			<td height="21" style="height:21px;">LIVE/DEAD fixable violet dead cell stain kit</td>
			<td>Life Technologies</td>
			<td>L34955</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transcription factor buffer set</td>
			<td>BD Pharmingen</td>
			<td>562725</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OneComp eBeads&#160;</td>
			<td>eBioscience</td>
			<td>01-1111-42</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">123 count eBeads</td>
			<td>eBioscience</td>
			<td>01-1234-42</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">CD45 AmCyan (clone 30-F11)</td>
			<td>BioLegend</td>
			<td>103138</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">CD3 PerCP-Cy5.5 (clone 17A2)</td>
			<td>BioLegend</td>
			<td>100218</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IL-17A FITC (clone TC11-18H10.1)</td>
			<td>BioLegend</td>
			<td>506910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IL-17F APC (clone 9D3.1C8)</td>
			<td>BioLegend</td>
			<td>517004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD4 APC-Cy7 (clone GK1.5)</td>
			<td>BD Biosciences</td>
			<td>560181</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">CD8 APC (clone 53-67)</td>
			<td>eBioscience</td>
			<td>17-0081-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T-bet PE-Cy7 (clone 4B10)</td>
			<td>BioLegend</td>
			<td>644823</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IFN&#947; FITC (clone XMG1.2)</td>
			<td>BD Biosciences</td>
			<td>557724</td>
			<td></td>
		</tr>
	</tbody>
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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 group have shown that hypertension is associated with an excessive accumulation of T cells in the vessels and kidney during the development of experimental hypertension6. Once in these tissues, T cells produce several cytokines that affect both vascular and renal function leading to vasoconstriction and sodium and water retention1,2. To fully understand how T cells cause cardiovascular and renal diseases, it is important to be able to identify and quantify the specific T cell subsets present in these tissues. T cell subsets are defined by a combination of surface markers, the cytokines they secrete, and the transcription factors they express. The complexity of the T cell population makes flow cytometry and intracellular staining an invaluable technique to dissect the phenotypes of the lymphocytes present in tissues. Here, we provide a detailed protocol to identify the surface and intracellular markers (cytokines and transcription factors) in T cells isolated from murine kidney, aorta and aortic draining lymph nodes in a model of angiotensin II induced hypertension. The following steps are described in detail: isolation of the tissues, generation of the single cell suspensions, ex vivo stimulation, fixation, permeabilization and staining. In addition, several fundamental principles of flow cytometric analyses including choosing the proper controls and appropriate gating strategies are discussed.

协议中所述的许可表面和细胞内标志物，从小鼠肾脏，主动脉及主动脉淋巴结在血管紧张素Ⅱ引起的高血压模型中分离的T细胞识别。代表性的结果介绍如下。 图1显示了用于从WT小鼠与血管紧张素II输注以诱导高血压的主动脉制得的单细胞悬液，以确定T细胞群体的门控策略。类似的策略在肾脏和淋巴结中。的前向?...

Watch this Scientific Journal Video about Intracellular Staining and Flow Cytometry to Identify Lymphocyte Subsets within Murine Aorta, Kidney and Lymph Nodes in a Model of Hypertension at JoVE.com

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

本文提供了详细的方法来识别和量化细胞内染色鼠肾，主动脉和淋巴结中存在功能性T淋巴细胞亚群和流式细胞仪。被选中的血管紧张素II诱导高血压模型来解释，一步一步的程序和流式细胞仪的基本原则和细胞内染色。

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 ...

Research

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Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
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Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

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鼠淋巴结基质细胞的分离

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The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

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There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

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Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

利用胶囊对生物达标中病毒样品的阴性染色

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most ...