Name: isolation of human umbilical vein endothelial cells and their use in the study of neutrophil transmigration under flow conditions
Uploaded: 2012-08-08T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions at JoVE.com

我们感谢的翩Colarusso博士和协助成像和图像分析活细胞成像设备;女士为她的技术援助Lailey和单位在提供人类脐带在卡尔加里，AB山麓医院5​​1。川崎病帕特尔博士是艾伯塔创新：健康解决方案的科学家。这项工作是从加拿大健康研究，并从加拿大创新和阿尔伯塔省科学和研究的权威机构的基础设备和基础设施补助机构的经营拨款支持。

1。人血管内皮细胞的分离和维护<ol><li>与人体血液和组织工作时，必须使用2级生物安全程序。用剪刀剪线从胎盘的血块和在分娩过程中夹紧电线造成的损害，然后仔细检查电线。切出并丢弃这些线部分。 </li><li>准备在1毫克/毫升，50毫升温线缓冲管，含有50毫克胶原酶注入新鲜的胶原酶溶液。 </li><li>变成无菌手套和消毒钝套管，电线钳，剪刀，三路停止旋塞，在层流罩纱布打开蒸压仪器托盘。 ...

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	<li>Nathan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+and+immunity:+challenges+and+opportunities.">Neutrophils and immunity: challenges and opportunities.</a> Nat. Rev. Immunol. 6, 173-182 (2006).</li><li>Diacovo, T. 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<html>研究人员经常使用不同血管床的血管内皮细胞研究中性粒细胞的招聘和轮回。例子包括，但不仅限于，真皮微血管内皮细胞，肝窦内皮细胞13和10人脐静脉内皮细胞。其中，人脐静脉内皮细胞获得了广受欢迎，因为它们相对容易隔离和可用性。 HUVEC的是一个强大的体外模型系统，可以模仿许多发生在体内的内皮进程。这个模型系统已帮助识别粘附分子和趋化因子?...

<table border="1"><tbody><tr><td> 试剂和设备 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>型胶原酶1 </td><td>沃辛顿</td><td> 4197 </td><td></td></tr><tr><td>线缓冲区</td><td></td><td></td><td>成分：140毫米氯化钠4毫米氯化钾10毫米，1毫米的NaH 2 PO 4 /的Na 2 HPO 4在pH 7.4的缓冲D-葡萄糖</td></tr><tr><td>内皮细胞介质（ECM） </td><td></td><td></td><td> M199与鹰盐辅以16％的人血清中含100单位的青霉素，链霉素100微克和0.3毫克L-glutamine/ml </td></tr><tr><td> M199 </td><td> GIBCO公司</td><td> 31100-035 </td><td></td></tr><tr><td>青霉素链霉素谷氨酸（100X） </td><td> Invitrogen公司</td><td> 10378-016 </td><td&gt; </td></tr><tr><td> ibidi商会</td><td> ibidi </td><td> 80606 </td><td></td></tr><tr><td>肿瘤坏死因子-α </td><td>预编程技术</td><td> 300-01A </td><td></td></tr><tr><td>人血白蛋白20％的解决方案</td><td>双子座生物制品</td><td> 800120050 </td><td></td></tr><tr><td> HBSS中没有的Ca 2 +和Mg 2 + </td><td>西格玛</td><td> H2487-10X </td><td></td></tr><tr><td> HBSS中使用的Ca 2 +和Mg 2 + </td><td>西格玛</td><td> H1387-10X </td><td></td></tr></tbody></table>

isolation of human umbilical vein endothelial cells and their use in the study of neutrophil transmigration under flow conditions

中性粒细胞白血细胞中最丰富的类型。它们形成了一个1的先天免疫系统的重要组成部分。在急性炎症，中性粒细胞的炎性细胞迁移到损伤部位。损伤部位的中性粒细胞的招聘是一个循序渐进的过程，包括第一，扩张血管，增加血流量;第二，微血管结构变化和血浆蛋白从血液中逸出;第三，滚动，粘附和对面的中性粒细胞的轮回内皮细胞;和第四积累的中性粒细胞在损伤2,3网站。宽阵列在体内和体外方法已发展到使这些过程的研究，4。这种方法侧重于在人类内皮细胞中性粒细胞轮回。 检查中性粒细胞的轮回中所涉及的分子过程的一个流行的方法是利用人Neutrophils相互作用与原代人脐静脉内皮细胞（HUVEC）5。中性粒细胞分离已直观地描述在别处6，因此本文将显示HUVEC的隔离方法。一旦分离，成长为汇合，内皮细胞被激活，产生粘附和活化分子的上调。例如，激活内皮细胞与细胞因子如TNF-α的结果增加了E-选择素和IL-8表达7。 E-选择素介导中性粒细胞和IL-8介导的激活和坚定的中性粒细胞粘附的捕获和滚动。后粘附中性粒细胞的轮回。轮回可以paracellularly发生，通过内皮细胞路口或transcellularly通过内皮细胞本身。在大多数情况下，这些相互作用的发现在血管7,8流条件下发生的。 平行板流动腔是一种广泛使用的系统，MI液力剪切应力话筒，发现在体内和体外 9,10水流条件下使中性粒细胞招聘研究。有几家公司生产平行平板流室和每个人都有优点和缺点。如果荧光成像是必要的，需要使用玻璃或光学类似的聚合物。内皮细胞不生长在玻璃上。 在这里，我们提出了一个简便，快速的中性粒细胞轮回相衬，DIC和荧光成像的方法，用低量ibidi通道幻灯片，支持快速的人血管内皮细胞的粘附和生长，并具有光学质量相媲美的聚合物制成的玻璃。在此方法中，内皮细胞生长和刺激在ibidiμslide。中性粒细胞被引入流量条件下，轮回进行了评估。路口旁程度与决心使实时荧光成像跨细胞轮回。

Watch this Scientific Journal Video about Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions at JoVE.com

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

本文首先介绍了人类脐静脉内皮细胞的分离的过程，然后演示如何使用这些细胞检查流量条件下的中性粒细胞轮回。通过使用低体积流量室从玻璃的光学特性的聚合物制成，活细胞荧光成像罕见的细胞群也是可能的。

分离人脐静脉内皮细胞和水流条件下其使用中的中性粒细胞轮回研究

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

Research

JoVE Journal

Immunology and Infection

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

量<em在体外</em&gt;激活的主人微血管内皮细胞在静态条件下测量的检测，以中性粒细胞粘附

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

科研

免疫与感染

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

一个流程粘附实验来研究白细胞招聘剪切应力的人力肝窦内皮细胞条件下

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Human Neutrophil Flow Chamber Adhesion Assay

人中性粒细胞流动室粘附实验

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

内皮细胞，细胞连接实时成像过程中中性粒细胞轮回在生理流动

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

在体外和体内模型来研究细菌粘附于血管壁下流动条件

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

流式细胞仪用来评估染色质的T细胞国家

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Quantification of Monocyte Transmigration and Foam Cell Formation from Individuals with Chronic Inflammatory Conditions

慢性炎症状态个体的单核细胞轮回和泡沫形成的定量研究

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Atherosclerosis, a leading cause of CAD, is initiated by the ...

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 ...

Simultaneous Study of the Recruitment of Monocyte Subpopulations Under Flow In Vitro

体外流动下单核细胞亚群的同时研究

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development ...