Name: tracking neutrophil intraluminal crawling, transendothelial migration and chemotaxis in tissue by intravital video microscopy
Uploaded: 2011-09-24T14:00:00
Description: Watch this Scientific Journal Video about Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy at JoVE.com

这项工作是支持由加拿大卫生研究院（CIHR，MOP - 86749）的研究资助。 L.刘CIHR新研究者奖（MSH - 95374）的收件人。

1。趋化琼脂糖凝胶的制备<ol><li>移取10 mL的2 × PBS 50 mL锥形管，并放进一个包含热水的烧杯中的热身管。 </li><li>吸取10毫升蒸馏水和另一50 mL锥形管中添加0.4克琼脂糖粉（其第略有松动）和热的混合物，直到刚刚在微波炉煮沸（在700瓦的微波炉〜1分钟） 。 </li><li>加入预热的2 × PBS琼脂糖溶液管，漩涡的混合溶液，并保持在温暖的热水的烧杯。 </li><li>微管的细胞趋化解决方案（例如，10μL0.5微克CX...

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	<li>Liu, L., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+leukocyte+recruitment:+organ-specific+mechanisms+of+action.">Molecular mechanisms of leukocyte recruitment: organ-specific mechanisms of action.</a> Thromb. Haemost. 89, 213-220 (2003).</li><li>Petri, B., Phillipson, M., Kubes, 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+physiology+of+leukocyte+recruitment:+an+in+vivo+perspective.">The physiology of leukocyte recruitment: an in vivo perspective.</a> J. Immunol. 180, 6439-6446 (2008).</li><li>Phillipson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraluminal+crawling+of+neutrophils+to+emigration+sites:+a+molecularly+distinct+process+from+adhesion+in+the+recruitment+cascade.">Intraluminal crawling of neutrophils to emigration sites: a molecularly distinct process from adhesion in the recruitment cascade.</a> J. Exp. Med. 203, 2569-2575 (2006).</li><li>Wong, C. H., Heit, B., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+regulators+of+leucocyte+chemotaxis+during+inflammation.">Molecular regulators of leucocyte chemotaxis during inflammation.</a> Cardiovasc. Res. 86, 183-191 (2010).</li><li>Gavins, F. N., Chatterjee, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+for+the+study+of+mouse+microcirculation+in+anti-inflammatory+drug+research:+focus+on+the+mesentery+and+cremaster+preparations.">Intravital microscopy for the study of mouse microcirculation in anti-inflammatory drug research: focus on the mesentery and cremaster preparations.</a> J. Pharmacol. Toxicol. Methods. 49, 1-14 (2004).</li><li>Bullen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic+imaging+techniques+for+drug+discovery.">Microscopic imaging techniques for drug discovery.</a> Nat. Rev. Drug Discov. 7, 54-67 (2008).</li><li>Hazelwood, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entering+the+Portal:+Understanding+the+Digital+Image+Recorded+Through+a+Microscope.">Entering the Portal: Understanding the Digital Image Recorded Through a Microscope.</a> Imaging Cellular and Molecular Biological Functions. , 3-43 (2007).</li><li>Hickey, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-selectin+facilitates+emigration+and+extravascular+locomotion+of+leukocytes+during+acute+inflammatory+responses+in+vivo.">L-selectin facilitates emigration and extravascular locomotion of leukocytes during acute inflammatory responses in vivo.</a> J. Immunol. 165, 7164-7170 (2000).</li><li>Cara, D. C., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+as+a+tool+for+studying+recruitment+and+chemotaxis.">Intravital microscopy as a tool for studying recruitment and chemotaxis.</a> Methods Mol. Biol. 239, 123-132 (2004).</li><li>Liu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LSP1+is+an+endothelial+gatekeeper+of+leukocyte+transendothelial+migration.">LSP1 is an endothelial gatekeeper of leukocyte transendothelial migration.</a> J. Exp. Med. 201, 409-418 (2005).</li><li>Heit, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI3K+accelerates,+but+is+not+required+for,+neutrophil+chemotaxis+to+fMLP.">PI3K accelerates, but is not required for, neutrophil chemotaxis to fMLP.</a> J. Cell Sci. 121, 205-214 (2008).</li></ol>

活体显微镜揭示炎症过程中白细胞招聘的细胞和分子机制的重要工具。半透明组织，如提睾肌及肠系膜微血管白细胞-内皮细胞相互作用的定量测定可视化仍然是应用技术1,5的黄金标准。传统的明活体显微镜具有许多独特的技术特点和在这些组织中透露的招聘机制是适用于大多数组织 在体内1,2,6。然而，在其他一些如肺，肝和脑组织的白细胞招聘机制已经提睾肌及肠系膜...

表：材料，特定试剂及设备<table border="1"><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>聚乙烯管，PE10 </td><td>碧迪</td><td> 427401 </td><td>编号0.28毫米×外径0.61毫米</td></tr><tr><td>墨汁</td><td> Speed​​ball艺术</td><td>超级黑</td><td> 100％的碳黑色颜料，没有染料</td></tr><tr><td>腐蚀</td><td>亚伦医疗</td><td> AA03 </td><td></td></tr><tr><td>甲苯噻嗪</td><td>拜耳医药保健，拜耳公司</td><td> DIN 02169592 </td><td></td></tr><tr><td>盐酸氯胺酮</td><td> Bioniche家畜保健加拿大公司</td><td> DIN 01989529 </td><td></td></tr><tr><td>小鼠重组，MIP - 2 </td><td> R＆D系统</td><td> 452 - M2 </td><td></td></tr><tr><td> WKYMVm </td><td>凤凰制药公司</td><td> 072-12 </td><td></td></tr><tr><td>琼脂糖</td><td> Invitrogen公司</td><td> 15510-027 </td><td>超纯</td></tr><tr><td>肝素</td><td>西格玛</td><td> H - 3393 </td><td></td></tr><tr><td>直立式显微镜</td><td>奥林巴斯</td><td> BX61WI </td><td></td></tr><tr><td> 3CCD彩色视频摄像机</td><td>索尼</td><td> DXC - 990 </td><td></td></tr><tr><td> HD - DVD录像机</td><td> LG电子公司</td><td> RH398H - M </td><td></td></tr><tr><td>电视监视器</td><td> LG电子公司</td><td> 22LG30 </td><td></td></tr><tr><td>水资源循环</td><td> Thermo Scientific的</td><td> HAAKE DC10 </td><td></td></tr><tr><td>蠕动泵</td><td>吉尔森;法玛西亚</td><td>吉尔森MINIPULS 3; Pharmacia公司的P - 3 </td><td></td></tr><tr><td>提睾肌板</td><td>萨斯喀彻温大学</td><td></td><td>自制</td></tr></table>

tracking neutrophil intraluminal crawling, transendothelial migration and chemotaxis in tissue by intravital video microscopy

招聘循环白细胞从血液流至发炎组织是一个重要和复杂的炎症 1,2的过程。在postcapillary静脉发炎组织，白细胞初步系绳上的小静脉壁壁面和滚动。滚动内皮细胞白细胞逮捕，并接受公司的附着力小静脉表面的趋化因子或其他趋化因子。搬迁许多粘附白细胞粘附在血管内皮细胞的交界外渗网站从最初的网站，这个过程被称为腔内爬行 3 。白细胞爬行，跨越内皮细胞（轮回）和血管外组织迁移朝来源的趋化因子（趋化）4。活体显微镜是一个强大的工具，在体内的白细胞-内皮细胞相互作用的可视化和细胞和分子机制揭示白细胞招聘2,5。在这份报告中，我们提供了一个全面的描述使用明活体显微镜，可视化，并确定在中性粒细胞在小鼠提睾肌的招聘的详细过程，中性粒细胞趋化梯度。为了诱导中性粒细胞的招聘，小块的琼脂糖凝胶（〜1毫米大小），其中包含中性粒细胞趋化MIP - 2（CXCL2，CXC趋化因子）或WKYMVm（色氨酸，赖氨酸，酪氨酸- VAL - D -蛋氨酸，一种人工合成的模拟细菌转肽）是放在毗邻postcapillary观察到的微静脉的肌肉组织。随着时间已过视频摄影和计算机软件ImageJ，内皮细胞，中性粒细胞transendothelial的迁移和组织中的迁移和趋化中性粒细胞腔内爬行可视化和跟踪。此协议允许腔内的爬行速度，轮回的时间，脱离时间，迁移速度，趋化速度，趋化指数在组织很多，如中性粒细胞的招聘参数，可靠和定量分析。我们证明，使用此协议，这些中性粒细胞招聘参数可以稳定的决心和方便地跟踪体内的单个细胞运动。

Watch this Scientific Journal Video about Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy at JoVE.com

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

我们描述了协议的明源在体内的中性粒细胞趋化中性粒细胞招聘时测量动态的中性粒细胞 - 内皮细胞相互作用的活体显微镜。中性粒细胞腔内爬行，transendothelial迁移和趋化鼠标提睾肌组织与时间已过视频摄影和ImageJ跟踪观察。

追踪中性粒细胞腔内的爬行，活体视频显微镜Transendothelial组织中的迁移和趋化

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

Research

JoVE Journal

Immunology and Infection

Neutrophil Extracellular Traps: How to Generate and Visualize Them

嗜中性粒细胞胞外的陷阱：如何生成和可视化

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them ...

科研

免疫与感染

Intravital Microscopy of the Inguinal Lymph Node

腹股沟淋巴结的活体显微镜

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

体外 共生分析评估病原体诱导中性粒细胞跨上皮迁移

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium

人中性粒细胞迁移的跨越培养膀胱上皮定量评价

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

生发中心的高度已解决活体条纹照明显微镜

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

在实时麻醉小鼠成像中性粒细胞和单核细胞在肠系膜静脉用活体显微镜

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice

Intravital Imaging of Neutrophil Priming Using IL-1ÃƒÅ½Ã‚Â² Promoter-driven DsRed Reporter Mice

使用IL-1β启动子驱动的红色荧光蛋白记者小鼠中性粒细胞引发的活体成像

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that ...

An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood

一种直接从一滴血液中快速中性粒细胞趋化分析的片上方法

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil ...

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

体外犬中性粒细胞胞外陷阱的形成: 荧光显微镜动态定量分析

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

双光子活体显微镜感病毒感染时气管黏膜的影像细胞相互作用

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...