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CXC趋化因子MIP - 2或12μL1毫米WKYMVm）含3μL墨汁1.5毫升的Eppendorf管的盖子，并混合使用微量的愿望（避免气泡）。 </li><li>切尖年底的200微升吸管尖和盖子微量110μL琼脂糖溶液（42℃），并立即拌匀，用另一个枪头（避免气泡）。 </li><li>趋化含凝胶存放在4 ° C。 </li></ol> 2。提睾肌的准备活体显微镜（图1） <ol><li>麻醉成年雄性小鼠腹腔注射10毫克/公斤甲苯噻嗪和200毫克/公斤盐酸氯胺酮的混合物。 </li><li>超过右颈外静脉和电动剃须刀的阴囊前方面的剃须面积。麻醉后，重要的是要给予特别的重视和关怀麻醉鼠标。可用于热灯，以防止低温鼠标。鼠标应无痛觉反射。 </li><li>设为横向切口，找到并采用充满100 U / ml肝素生理盐水的PE - 10管导尿颈静脉。颈内静脉插管在必要时需要额外的麻醉剂和药品的管理。 </li><li>修正脐磁带鼠标躺在自制的提睾肌板（图1）面对老鼠的后腿。 </li><li>将电路板连接到37℃水的循环，提睾肌和鼠标保持身体温暖。 </li></ol>注意:从2.6到2.14之间的所有的程序必须非常轻柔5。 <ol start="6"><li>作出在阴囊皮肤切口，显露左提睾肌。仔细剖析相关的筋膜肌肉。 </li><li> Superfuse提睾肌与37 ° C -回暖碳酸氢盐缓冲液（131.9氯化钠，氯化钾4.7，1.2 硫酸镁 ，20碳酸氢钠3，在MM，pH值7.4），使用蠕动泵。 </li><li>领带4-0缝合举行清晰地收看提睾肌板的玻璃底座上的提睾肌的末端。 </li><li>纵向烧灼的提睾肌。用4-0缝合，保持肌肉平坦，沿基座的边缘安全。从底层肌肉的睾丸和附睾分离，并移动到腹腔。 </li><li> Superfuse〜0.6毫升/分钟，用37℃温灌注缓冲和暴露的肌肉覆盖了22 × 22毫米的玻璃盖玻片的肌肉。 </li><li>提睾肌板，放在显微镜阶段研究的肌肉，在显微镜下，找到一个合适的postcapillary的微静脉（选择直，分枝和正常的剪切速率和直径在25-40微米的微静脉）和调整摄像头使小静脉，要么向左或向右端的电视监视器上的垂直位置的可视化。 </li><li>经过30分钟的平衡，记录选定的5分钟为基准控制数据使用录像机postcapillary的小静脉视频图像。 </li><li>停止灌流，取出盖玻片上的肌肉。 </li><li>提睾肌在350微米预选区的表面上放置一个〜1毫米大小的趋化因子含有凝胶和观察postcapillary微静脉平行，加盖玻片举行的地方凝胶superfuse的肌肉组织，在一个非常速度慢（≤10μL/分钟），以便建立一个缓慢释放和凝胶形成的趋化因子梯度。 </li><li>记录的趋化因子含有凝胶后90分钟的视频图像。在录制过程中，调整和保持显微镜上坚持重点，爬行，transmigrating chemotaxing的小静脉内和肌肉组织中的白细胞。 </li><li>实验后，导入的视频文件到计算机进行分析。 </li></ol> 3。细胞示踪使用ImageJ <ol><li>在计算机上，提取和视频转换为AVI格式（例如，免费使用的计算机的软件bitRipper，DVD视频转换为AVI文件）。 </li><li>使用视频编辑软件生成的时间，失效的电影。例如，使用Windows Movie Maker使一个失效时间的电影（1 / 512或1 / 1024时间推移一个T 30 fps的速率）从原来的，实时视频。 DV - AVI格式转换和保存时间已过未压缩的电影。 </li><li>校准微米的图像记录在相同的显微镜设置，导入到计算机的图像，打开ImageJ的图像。 ImageJ，总像素显示屏幕（例如，720 × 480像素）的左上角。测量屏幕的大小，在X和Y轴（例如，200 × 150微米）。由此看来，计算每微米的像素（例如，X =二百分之七百二十〇= 3.6像素/微米，和Y =一百五十○分之四百八十零= 3.2像素/微米）的数量。 </li><li>要导入的电影，再次打开ImageJ，点击"文件 - 导入使用QuickTime电影插件"，选择要分析的电影，然后单击"确定"，"QT电影开瓶器"界面。 </li><li>点击"插件手动跟踪"来跟踪细胞。在填写有关信息，在底部前开始跟踪的领域。简单地说， <ol><li>时间间隔（秒）= fold-time-lapse/30（例如，1020 ×时间推移将三十〇分之一千零二十零= 34秒/帧的时间间隔）。 </li><li> x / y的校准=微米/像素测量使用的千分尺的形象校准。 </li><li>于校准= 0（鼠标提睾肌是一层极薄的组织和细胞爬行和迁移，大约是在明场透2D）。 </li><li>搜索中心= 1平方米大小​​。 </li><li>点尺寸/线宽/线字体大小:如果需要，可以调整。 </li></ol></li><li>选择一个稳定和清晰的点作为参考点。该参考点可以是任何明确的和小的结构保持不变，在整个​​实验的稳定。点击"添加轨道"来跟踪从第一参考点到最后一帧，并单击"结束曲目"。数据自动出现在结果表。 </li><li>轨道爬行和迁移的中性粒细胞逐个点击"添加轨道"来跟踪从它的外观在组织细胞在每帧的消失，单击"结束追踪"完成并保存在Microsoft Excel中的结果。 </li></ol> 4。中性粒细胞招募参数分析<ol><li>打开在Microsoft Excel中的结果文件，并分析数据（分析，考虑到参考点的变化）。 </li><li>腔内爬行<ol><li>爬行距离:总距离的细胞爬壁，从最初的网站在管腔内的最优轮回网站（微米）。 </li><li>爬行速度:爬行距离/时间（微米/分钟）。 </li></ol></li><li> Transendothelial迁移<ol><li>轮回时间:从细胞开始的时间轮回跨内皮细胞的时候，当整个胞体外面的小静脉，可以看到在流明（分钟或秒）和无细胞体。 </li><li>支队时间:从整个胞体外面的小静脉（后立即轮回）的时间点时，细胞失去接触与微静脉（尾缩回）（分钟或秒）。 </li></ol></li><li>趋化作用，在组织<ol><li>迁移距离:距离的细胞从起点移动到组织中迁移的终点（微米）的总和。 </li><li>移动速度:在组织/时间的迁移距离（微米/分钟）。 </li><li>趋距离:距离细胞的总和，在组织中的X轴（微米）的迁移。 </li><li>趋化速度:趋化距离/时间（微米/分钟）。 </li><li>趋化指数:组织中的迁移距离除以趋化距离的比例。 </li></ol></li></ol> 5。代表性的成果: 虽然bightfield活体显微镜用于白细胞 - 内皮细胞相互作用的研究，未必一定中性粒细胞，我们确认，我们的组织学研究中，超过95％的招募细胞中的中性粒细胞趋化处理的提睾肌的肌肉确实是中性粒细胞。在这份报告中，使用选择性中性粒细胞趋化因子，我们目前跟踪的中性粒细胞在体内的招聘程序。具体来说，我们描述了一个跟踪腔内的中性粒细胞爬行，transendothelial迁移，并在睾使用时间推移活体视频显微镜和ImageJ麻醉小鼠的肌肉组织的趋化作用的协议。缓慢释放的趋化含琼脂糖凝胶上的提睾肌细胞趋化，并允许在组织成立的一个趋化因子梯度。嗜中性粒细胞趋化因子诱导中性粒细胞在小鼠提睾postcapillary静脉内皮细胞间的相互作用。整个实验是一个堂堂正正的明预计一个彩色视频摄像机到电视监视器和录像机记录的视频图像的活体显微镜下可视化。我们确定的中性粒细胞腔内爬行，transendothelial迁移和迁移响应和肌肉组织中趋化中性粒细胞趋化因子MIP - 2和琼脂糖凝胶WKYMVm在准备（图2）。我们发现，MIP - 2（0.5微米）和WKYMVm（0.1毫米）在类似的速度，中性粒细胞transendothelial的迁移和比较长的时间，中性粒细胞迁移和肌肉组织中趋化小静脉支队引起中性粒细胞几乎腔内爬行相同的速度和类似的中性粒细胞趋化指数（P&gt; 0.05，Student t检验）。 <img src="/files/ftp_upload/3296/3296fig1.jpg" alt="图1" /> 图1活体显微镜系统的示意图。鼠标提睾肌exteriorized清晰地收看基座上的提睾肌板在显微镜阶段，并与37 ° C加热碳酸氢盐缓冲液灌流。直立的显微镜连接为明活体显微镜与CCD彩色视频摄像机。也是一个单色深冷CCD数码相机连接到显微镜，荧光活体显微镜端口，图像直接通过计算机处理。 <img src="/files/ftp_upload/3296/3296fig2.jpg" alt="图2" /> 图2。中性粒细胞招聘明活体显微镜参数。招募中性粒细胞诱导的中性粒细胞趋化的逐步释放，MIP - 2在琼脂糖凝胶法制备的WKYMVm放置350微米，相邻postcapillary微静脉。时间失效的视频数据进行分析处理后的实时视频录制的实验由ImageJ。中性粒细胞腔内爬行（一），轮回的时间和脱离时间（B），在组织中的迁移速度和趋化速度（三），提睾肌（四）趋化指数测定后，MIP - 2或WKYMVm琼脂糖凝胶的管理提睾肌在C57BL / 6小鼠（N = 3，= 22（A和B）和27（C和D）分别为MIP - 2，和= 26（A和B）和44（＃跟踪细胞在C和D）分别为WKYMVm）。

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没有利益冲突的声明。

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 1显示的完全不同。此外，荧光显微镜已被用于一些不太透明的组织。 明活体显微镜光损伤，以活体细胞的功能，这是已知的荧光显微镜相比，更多的生理和危害较小的细胞和组织（因此用较少的文物）时，长时间的成像观察动态的细胞行为活的动物是必要的。这也更方便，成本更低，没有荧光标记。另一方面，在活体显微镜与透射成像，自动跟踪的细胞运动是不可能的，目前市场上的商业成像软件。但是，它是很容易的建立一个独立的活体荧光成像系统（例如，通过投影图像到荧光的CCD相机和计算机）在同明活体显微镜（图1）。这明显微镜和荧光成像之间切换，在一个单一的实验相同的样品制备提供了方便。这也使得它可以自动跟踪，荧光活体显微镜下运动时，荧光标记的细胞标记的细胞和背景是足够大的和合适的成像软件的荧光强度之间的对比是在计算机上安装。 如我们这里的介绍描述，我们展示了这种在体内中性粒细胞招聘的全过程的直接观察和确定每一个招聘步骤中的细胞和分子的功能的技术价值。随着趋化包含在琼脂糖凝胶的制备和组织举行的，单向的趋化梯度可以建立在组织中引起白细胞招聘反应类似自然发生在局部炎症8,9。定向运动走向趋化源postcapillary微静脉白细胞可以清楚地明活体显微镜和视频摄影可视化。随着时间推移视频处理，细胞运动可以由ImageJ跟踪和一系列高度重复性的参数可以衡量。与特定的转基因小鼠，抑制剂和选择性的趋化因子，检测系统，帮助我们揭示白细胞招聘特定蛋白质的功能。例如，这种技术帮助我们确定的中性粒细胞transendothelial迁移的调节10作为看门人血管内皮细胞的作用，为LSP1，在中性粒细胞腔内爬行（αMβ2整合）的Mac - 1的作用，为最佳transendothelial迁移的重要一步3，组织11中性粒细胞趋化PI3Kγ的作用。

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 ...

tracking-neutrophil-intraluminal-crawling-transendothelial-migration

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

19.6K 次观看.  University of Saskatchewan. 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。

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

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria1, fungi2 and parasites3. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent4. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase5. 
Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility6.
We describe methods to isolate Neutrophil Granulocytes from peripheral human blood7 and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.

Neutrophil Extracellular Traps (NETs) are an important innate immune mechanism to fight pathogenic bacteria, fungi and parasites. Here we describe methods to isolate neutrophil granulocytes from human blood and to activate them to form NETs. We present preparation techniques to visualize NETs in light and electron microscopy.

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

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 response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

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 ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

活体成像的鼠标使用2 - 光子显微镜的胸腺

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

活细胞视频显微镜病病原菌的吞噬作用

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

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

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

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

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

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

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. During infection of the urinary bladder, polymorphonuclear leukocytes (PMN; neutrophils) migrate from the bloodstream and traverse the bladder epithelium. Failure to resolve infection in the absence of a neutrophilic response demonstrates the importance of PMN in bladder defense. To facilitate colonization of the bladder epithelium, uropathogenic Escherichia coli (UPEC), the causative agent of the majority of urinary tract infections (UTIs), dampen the acute inflammatory response using a variety of partially defined mechanisms. To further investigate the interplay between host and bacterial pathogen, we developed an in vitro model of this aspect of the innate immune response to UPEC. In the transuroepithelial neutrophil migration assay, a variation on the Boyden chamber, cultured bladder epithelial cells are grown to confluence on the underside of a permeable support. PMN are isolated from human venous blood and are applied to the basolateral side of the bladder epithelial cell layers. PMN migration representing the physiologically relevant basolateral-to-apical direction in response to bacterial infection or chemoattractant molecules is enumerated using a hemocytometer. This model can be used to investigate interactions between UPEC and eukaryotic cells as well as to interrogate the molecular requirements for the traversal of bladder epithelia by PMN. The transuroepithelial neutrophil migration model will further our understanding of the initial inflammatory response to UPEC in the bladder.

We developed an in vitro model that mimics an important component of the acute inflammatory response during infection of the bladder with uropathogenic Escherichia coli. The transuroepithelial neutrophil migration assay enables quantitative assessment of human neutrophil migration across bladder epithelia, cultured on permeable supports, in response to bacterial infection or chemoattractant substances.

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

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

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 ...

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

在Mesenterial静脉的活体显微镜白细胞，内皮细胞和血小板白细胞相互作用小鼠

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

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

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

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

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal &#8212; used as a measure of priming 2) in vivo neutrophil labeling &#8212; achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models.

This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.

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 ...