This work was supported by EMBO (to Y.E.), Foundation Machaon (to Y.E.) and SNSF (to B.A.I.). We thank the Bioimaging Core Facility for the availability of the Nikon A1r microscope and technical assistance. We thank Mrs. Clarissa Bartley for English correction.

注:动物的程序是按照动物护理在瑞士日内瓦的机构伦理委员会和州兽医办公室进行。授权号GE / 63/14 1.准备一个单细胞悬液，从骨髓<ol><li>牺牲小鼠（8-12周龄）颈椎脱位。消毒后腿以70％的乙醇。从后腿去除皮肤。 </li><li>解剖出鼠标的股骨和胫骨和腿部用手术刀去除组织。冲洗的腿与90％的乙醇和地方变成培养皿填充有PBS。 </li><li>根据培养罩，干净的纸巾，从股骨和胫骨用手术刀和独立的膝关节。切断骨头的两端。 </li><li>从每个骨骼使用23G针和1ml注射器填充有制备培养基（无酚红的DMEM / F12，1％大鼠血清），到50毫升冲洗骨髓的螺旋管。通过经由23Gね微妙传代破坏细胞聚集体edle和1ml注射器。把总体积25毫升的准备中。 </li><li>离心机250 XG，5分钟，4℃。弃上清。在1ml重悬沉淀红细胞溶解RBLC缓冲液（8.3克氯化铵 ，1克碳酸氢钠，加入1ml EDTA（100毫米），完整到1L蒸馏水，过滤0.22微米）在15ml的螺旋管。孵育30秒在冰上。 </li><li>加入10毫升制备培养基和离心机250×g离心5分钟，4℃。弃上清，悬浮在2毫升的准备中。 </li></ol> 2.中性粒细胞富集阴性选择<ol><li>加入150微升鼠标中性粒细胞富集的鸡尾酒（细胞分离试剂盒的平台，如EasySep）。混合并在冰上孵育10分钟。 </li><li>加入10毫升酚红的DMEM。倒置一次和离心机250×g离心3分钟，4℃。 </li><li>弃上清。悬浮颗粒在1.75毫升制备介质在5毫升聚苯乙烯圆底管。加入250微升比奥在选择鸡尾酒。混合并在冰上孵育10分钟。 </li><li>涡磁性粒子（从细胞分离平台盒）30秒重悬颗粒。加入500μl磁性粒子的细胞悬浮液。混合并在冰上孵育10分钟。 </li><li>将管插入磁铁。等待3分钟。 </li><li>反转磁铁到一个新的5毫升聚苯乙烯圆底管，收集所需的未标记细胞。不要把最后一滴挂在管。不需要的细胞将保留在磁铁内部的管。丢弃该管成生物危险废物。 </li><li>将新管插入磁体。等待3分钟。 </li><li>反转磁铁到新款15毫升螺旋管，收集所需的未标记细胞。不要把最后一滴挂在管。 </li><li>完整体积至5ml用酚红的DMEM。 </li></ol> 3.标签中性粒细胞<ol><li>添加0.5微升橙色染料如细胞跟踪橙色（工作浓度为1μM，CMRA - 氯甲基rodol乙酸酯，库存的10mM在DMSO中）。在37℃下2分钟。 </li><li>加入2.5毫升的准备中。离心机250 XG，5分钟，4℃。 </li><li>弃上清。重悬沉淀在1ml制备介质在1.5ml管中。 </li><li>以等分用血细胞计数器计数。 </li><li>孵育5分钟，在37℃。离心机250 XG，5分钟，4℃。 </li><li>弃上清。重悬沉淀在1ml PBS中。离心机250×g离心5分钟，4℃下洗。 </li><li>弃上清。悬浮颗粒每100微升PBS 10×10 6个细胞 。置于冰上，直到静脉注射。注:电池必须IV尽快注射越好。 6-12x10 6中性粒细胞/鼠标，通常获得的。 </li></ol> 4.鼠标准备活体显微镜注意:步骤4.1）和4.2）应在实验开始时进行，以避免对IC标记的嗜中性粒细胞的延长的等待时间即<ol><li>称量CX 3 CR1 GFP小鼠（8-12周龄）。 </li><li>制备800微升麻醉药（氯胺酮60毫克/公斤，甲苯噻嗪4.5毫克/千克，acepromazone 1.75毫克/公斤的PBS）。 </li><li>麻醉鼠标。注射IP用200微升/ 20克鼠标。按捏脚趾并通过检查呼吸频率控制麻醉。 </li><li>注入的中性粒细胞（10×10 6个细胞在100μlPBS中）静脉内使用的侧面尾静脉或眶后窦，在实验者的便利性。 </li><li>把鼠标放在加热垫，保护眼睛，眼部凝胶。 </li><li>的玻璃盖玻片（直径为35毫米）放置在10厘米组织培养皿，它有一个直径30毫米的孔。使用油是维持组织培养皿中的盖玻片接触。 </li><li>切开腹部皮肤用剪刀，露出腹膜。腹膜切开用剪刀，露出肠。 </li><li>加入200μl的PBS（预加热，在37℃）到peritoneal腔。拿鼠标在你的手，把它面朝下，使肠道得到了腹腔中有一个温和的压力。鼠标放置在组织培养皿。 </li><li>轻轻deroll用消毒棉签将肠道到盖玻片，以暴露肠系膜血管。 </li><li>切纸组织的乐队，与37℃加热PBS弄湿。固定与面纸的部分小肠，以减少从肠蠕动而产生的运动。 </li><li>把组织培养皿和鼠标37℃恒温室内部到倒置显微镜的定做托盘阶段。引入含有麻醉药SC到后腿的注射器。注:此外，鼠标可以接收氧气（0.5升/分钟）用的掩模。 </li><li>控制鼠标麻醉通过脚趾捏和检查呼吸频率每30分钟。如果需要，提供麻醉药的提升（20微升）妥善维持麻醉。不E:应避免脉管的干燥。因此，它的湿度是通过定期润湿用于固定肠用37℃温热的PBS的纸巾保持。 </li></ol> 5.测量中性粒细胞和单核细胞行为发炎的血管在体内使用活体显微镜<ol><li>设置激光器488-和561-在需要避免激光诱导光毒性最低的功耗。在我们的实验中，这是低于0.5％。绿色荧光蛋白在单核细胞和橙色染料中性粒细胞的强度是如此明亮，通常为0.3％就足够了。注意:此表示0.15〜0.25毫瓦与我们的系统的激光功率。 </li><li>收购的512×512像素（ 即 635微米）的2.2秒的图像，使用倒置显微镜的20X干客观开放的针孔。使用10的Z深度20微米，以确保足够的信号与低功率的激光。 注:我们通常用做实验用12至16的Z深度56，M。在呈现附图和电影的情况下，在z深度为20微米。相衬允许内皮墙壁的识别和成像肠系膜上静脉进行。由于巡逻细胞移动相当缓慢，5-10微米/分钟6的速度，记录的关注的一个领域，每20秒令人满意。所描述的设置，电动阶段允许感兴趣多达7字段的同时记录。 </li><li>鼠标放置在显微镜的恒温室中的肠系膜血管被允许他们从获取的第一部电影的前准备30分钟恢复。在此期间，选择感兴趣的几个领域。重点放在最接近目标容器的腔表面。如果需要，采取轻微的自体荧光血管内皮细胞的优势，设置焦点。 注:鼠标准备在手术过程中稍微强调内皮。因此，轧制中性粒细胞和/或单核细胞所用的第一15分钟以下鼠标制剂中观察到。 </li><li>录制的第一部电影稳态条件下30分钟，一张图片，每20秒。 注:该软件获取的每个感兴趣字段在2.2秒和电动阶段自动地从一个场移动到另一个。这又回到了在20秒内的第一个位置，在记录电影的30分钟。 </li><li>要启动血管炎症，加入20微升PBS含100微克TLR2的下降/ TLR1激动剂Pam3CSK4 12直接到成像的船只。 </li><li>控制聚焦为每个感兴趣的领域。 注:在收购的过程中，总有重点的对Z轴的细微变化。尽管10-20微米的z深度，这会导致降低的荧光强度的。因此，手动重新集中的每个感兴趣字段在每部电影的最后，如果需要的话。 30分钟时段和最多3个小时，拍摄电影TAL炎症开始后。 </li><li>在实验结束时，通过颈脱位牺牲麻醉小鼠。 </li></ol> 6.代电影文件和白细胞跟踪注:尼康采集软件生成.nd2文件，但下面的程序将与其他软件和品牌的同类。 <ol><li>文件导出为TIFF系列。 </li><li>转到ImageJ的软件<a href="http://rsb.info.nih.gov/ij/">（http://rsb.info.nih.gov/ij/）。</a>导入TIFF系列。每个信道为不同的文件。 </li><li>具有2.0像素为绿色和红色通道，以减少背景噪声（处理过程&gt;滤波器&gt;中位...）的半径应用中值滤波器。 </li><li>文件转换成二进制（流程&gt;二进制&gt;进行二进制）。启用软件来计算阈值的每个图像。 </li><li>合并频道合并为一个图像序列（图像&gt;彩色&gt;合并通道...）。选择格力的单核细胞n个信道，在红色通道，并在灰色通道的透射光的中性粒细胞。 </li><li>堆叠图像转换为RGB颜色（图像&gt;颜色&gt;堆栈RGB）。 </li><li>文件保存为.AVI </li><li>数据被导入并在了Imaris软件（位平面）编制。单核细胞和中性粒细胞的运动即期跟踪向导，然后跟踪。 </li></ol>

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The authors declare that they have no competing financial interests.

在这个手稿中描述的方法提供了一个一致的方法来有效地研究单核细胞和中性粒细胞行为的稳态和炎症的条件下肠系膜静脉。 制剂的关键步骤是用PBS-湿的薄纸肠的固定。如果执行得当，肠系膜血管是很好的暴露在盖玻片进行图像采集。这使得感兴趣的几个领域的选择，监测肠系膜静脉白细胞行为。 我们的方案涉及肠系膜血管的外化的实验开始前，使（1?...

<html>嗜中性粒细胞和单核细胞是先天免疫系统，它在血液中循环连续的细胞。在损伤或感染，炎症信号诱导白细胞血细胞渗出到受损和感染的组织中，本文引发细胞免疫应答1-3。白细胞动员的快速性决定了免疫应答的积极成果。这些复杂的过程依赖于特定的分子 （如选择素，血管内皮细胞结合的趋化因子）出现在发炎的内皮细胞，帮助循环白细胞和血管内皮细胞1-3之间建立接触胶。要得到牵连白细胞分子见解招募级联，可视化细胞募集的动力学和跟踪每个小区/群体的行为是重要的。一种有效的方法包括在表达荧光蛋白在感兴趣的细胞的转基因小鼠活体显微镜。 内容"&gt;迄今为止，使用活体显微镜几种方法被开发以图像脉管4,5，例如，耳真皮脉管或肠系膜静脉由活体共聚焦显微镜成像被用来确定的鼠的Ly6C 低的单核细胞和人巡逻行为CD14 变暗 CD16 +单核细胞上的血管的稳定状态条件下6-8腔侧，鼠标提睾脉管模型通常用于监测炎症或缺血性病症中的转基因小鼠嗜中性粒细胞之下或炎症的Ly6C 高的单核细胞的行为。在这情况下，提睾通过阴囊内注射IL1β，CCL2，TNFβ或fMLP的刺激后2-4小时，组织，然后手术形象化和活体共聚焦显微镜9-11分析。 以下方案描述了一种方法来监控单核细胞和中性粒细胞的同时与任何倒置荧光共聚焦显微镜。此外，我们的方法，详细介绍了如何前映像相同的容器（稳态条件）和炎症后如何遵循白细胞募集的动力学。为此目的，我们使用在CX 3 CR1 GFP /重量鼠标，其单核细胞表达eGFP的，静脉注射一种橙色色素标记的鼠中性粒细胞。使用倒置共聚焦显微镜，它是可能的（1）来跟踪和分析稳态条件和（2）以符合两个单核细胞和中性粒细胞在同一容器中的后局部炎症招募下巡逻的Ly6C 低的单核细胞。在这里，我们创建了炎症的使用TLR2 / TLR1激动剂Pam3CSK4 12。而且，这样的成像可有助于确定是否对特定敲除小鼠或在阻断抗体6,9,13的存在下进行的，在各个步骤的白细胞募集级联感兴趣的特定分子的作用。

<table><tbody><tr><td>5 ml polystyrene round bottom tubes&amp;nbsp;</td><td>Beckton Dickinson</td><td>352058</td><td></td></tr><tr><td>10x CFI Plan Apochromat 0.45&amp;nbsp;&amp;nbsp; DT:4mm&amp;nbsp;</td><td>Nikon</td><td></td><td></td></tr><tr><td>20x CFI Plan Apochromat VC 0.75 DT:1mm&amp;nbsp;</td><td>Nikon</td><td></td><td></td></tr><tr><td>Cell Tracker Orange CMRA Dye</td><td>Life Technologies</td><td>C34551</td><td></td></tr><tr><td>EasySep Magnet</td><td>Stem Cell Technologies</td><td>18000</td><td></td></tr><tr><td>EasySep Mouse Neutrophil Enrichement kit</td><td>Stem Cell Technologies</td><td>19762</td><td></td></tr><tr><td>EDTA</td><td>Sigma Aldrich</td><td>E6758</td><td></td></tr><tr><td>FCS</td><td>PAA</td><td>A15-042</td><td></td></tr><tr><td>Immersion Oil Type A</td><td>Nikon</td><td></td><td>any viscous oil&amp;nbsp;</td></tr><tr><td>Life Box Temperature Control System</td><td>Life Imaging</td><td></td><td></td></tr><tr><td>NaHCO3</td><td>Sigma Aldrich</td><td>S5761</td><td></td></tr><tr><td>NH4Cl</td><td>Sigma Aldrich</td><td>A9434</td><td></td></tr><tr><td>Nikon A1R confocal microscope</td><td>Nikon</td><td>A1R</td><td>inverted microscope, motorized x/y/z stage, NIS elements software</td></tr><tr><td>PBS</td><td>Life Technologies</td><td>D8537</td><td></td></tr><tr><td>phenol red free DMEM/F12</td><td>Life Technologies</td><td>21041-025</td><td>any phenol red free medium is suitable</td></tr><tr><td>PAM3CSK4</td><td>Invivogen</td><td>tlrl-pms</td><td>reconstitute in PBS</td></tr><tr><td>Rat serum</td><td>Stem Cell Technologies</td><td></td><td>included in EasySep Mouse Neutrophil Enrichement kit</td></tr><tr><td>Tissue culture dish 100</td><td>TPP</td><td>93100</td><td></td></tr></tbody></table>

imaging neutrophils and monocytes in mesenteric veins by intravital microscopy on anaesthetized mice in real time

有效的免疫反应是依赖于血液中的白细胞的快速动员到感染或损伤的部位。 调查白细胞迁移体内对于理解白细胞跨内皮迁移和相互作用与血管内皮的分子基础是至关重要的。一种有效的方法包括在表达荧光蛋白在感兴趣的细胞转基因小鼠活体显微镜。 在这里，我们提出了一个协议，用于成像的单核细胞和中性粒细胞在CX 3 CR1 GFP /鼠标重量静脉注射橙色染料标记的嗜中性粒细胞用倒置的共聚焦显微镜。时间推移电影收集从30分钟到几个小时的成像允许的白细胞行为在肠系膜静脉两个稳态和炎性条件下分析。我们还描述的步骤，以在本地引起血管炎症TLR2 / TLR1激动剂Pam3SK4和监控subsequeNT招聘中性粒细胞和单核细胞。 所提出的技术也可以用于监测白细胞的其他人口和调查使用等刺激或转基因小鼠的分子参与了白细胞招聘或贩运。

手稿描述了一种优化的协议来方便地监视单核细胞和嗜中性粒细胞中的实时麻醉的小鼠的肠系膜静脉的行为。使用37℃-恒温室的是强制性的，以保持鼠标的温度，也由于白细胞的温度相关的运动。鼠标的制备显示于图1中。图2示出了所有在显微镜下看到的面积。透射光使肠系膜静脉（红色箭头）和动脉（蓝色箭头）的标识。 治疗ImageJ的软件生成的视?...

Watch this Scientific Journal Video about Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time at JoVE.com

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

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 ...

imaging-neutrophils-monocytes-mesenteric-veins-intravital-microscopy

Research

JoVE Journal

Immunology and Infection

10.4K 次观看.  University of Geneva. 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.

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

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

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling3. Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)4. Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment2,5. In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm3 size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo.

We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.

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 ...

科研

免疫与感染

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

在活体小鼠的血管炎症和血栓形成的异型血小板中性粒细胞被激活的内皮细胞相互作用对实时成像

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

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 ...

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 ...

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

股骨窗口商会模型<em&gt;在体内</em在小鼠骨髓&gt;手机跟踪

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic stenosis. Vascular surgery is a viable option in selected cases, but for patients without indications for surgery such as progression to rest pain, critical limb ischemia, or major disruptions to life or work, there are few possibilities for mitigating their disease. Cell therapy via monocyte-enhanced perfusion through the stimulation of collateral formation is one of a few non-invasive options.
Our group examines arteriogenesis after monocyte transplantation into mice using the hindlimb ischemia model. Previously, we have demonstrated improvement in hindlimb perfusion using tetanus-stimulated syngeneic monocyte transplantation. In addition to the effects on the collateral formation, tumor growth could be affected by this therapy as well. To investigate these effects, we use a basement membrane-like matrix mouse model by injecting the extracellular matrix of the Engelbreth-Holm-Swarm sarcoma into the flank of the mouse, after occlusion of the femoral artery.
After the artificial tumor studies, we use intravital microscopy to study in vivo tumor-angiogenesis and monocyte homing within collateral arteries. Previous studies have described the histological examination of animal models, which presupposes subsequent analysis to post-mortem artifacts. Our approach visualizes monocyte homing to areas of collateralization in real time sequences, is easy to perform, and investigates the process of arteriogenesis and tumor angiogenesis in vivo.

Monocytes are important mediators of arteriogenesis in the context of peripheral arterial disease. Using a basement membrane-like matrix and intravital microscopy, this protocol investigates monocyte homing and tumor-related angiogenesis after monocyte injection in the femoral artery ligation murine model.

小鼠外周动脉疾病模型中单核细胞归巢和肿瘤相关血管生成的活体显微镜观察

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic ...

医学

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;

毛里小肠内皮淋巴细胞的内质成像

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal ...

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery.
After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

多光子活体成像用于监测小鼠后肢模型中动脉生成过程中白细胞募集

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...

<meta charset="utf8"></head><body>内皮 PAR1 在 I/R 损伤期间白细胞粘附和 NET 形成中的作用

Visualization of Neutrophil Extracellular Traps in Mesenteric Venules After Mesenteric Ischemia-Reperfusion Injury via Intravital Microscopy

Intestinal ischemia-reperfusion (I/R) injury is an acute condition characterized by tissue damage resulting from restricted blood flow to the mesenteric vessels, leading to both local and systemic pathologies with a poor prognosis. Both ischemia and reperfusion trigger a series of cellular and molecular responses, with inflammatory cells serving as key regulators of the pathology. These interactions with the ischemic endothelium are mediated by multiple adhesion receptors. Several animal models have been established to mimic this pathology and investigate the involved molecular pathways. In this study, a microsurgical model of I/R injury is combined with intravital microscopy to visualize leukocyte rolling, adhesion, and neutrophil extracellular trap (NET) formation. This model is applied to transgenic mice deficient in endothelial PAR1 (F2r) to assess the impact of PAR1 on leukocyte rolling and NET formation 1 h after ischemia and immediately following reperfusion. In vivo, Acridine Orange leukocyte staining was employed, and NETs were visualized using a nucleic acid stain. Interestingly, reduced leukocyte adhesion and NET formation were observed in mice lacking the endothelial PAR1 receptor. This model enables the in vivo analysis of key regulators involved in I/R injury.

<meta charset="utf8"></head><body>通过活体显微镜观察肠系膜缺血再灌注损伤后肠系膜小静脉中中性粒细胞细胞外陷阱的可视化

This protocol presents a method for inducing mesenteric ischemia-reperfusion injury and visualizing neutrophil extracellular traps (NETs) in mesenteric venules using intravital microscopy. Blood flow in the superior mesenteric artery was restricted for 1 h, followed by reperfusion, allowing direct quantification of leukocyte adhesion and NETosis.

通过活体显微镜观察肠系膜缺血再灌注损伤后肠系膜小静脉中中性粒细胞细胞外陷阱的可视化

Intestinal ischemia-reperfusion (I/R) injury is an acute condition characterized by tissue damage resulting from restricted blood flow to the mesenteric ...

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

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此视频中的章节

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

在活体小鼠的血管炎症和血栓形成的异型血小板中性粒细胞被激活的内皮细胞相互作用对实时成像

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

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

股骨窗口商会模型

小鼠外周动脉疾病模型中单核细胞归巢和肿瘤相关血管生成的活体显微镜观察

毛里小肠内皮淋巴细胞的内质成像

多光子活体成像用于监测小鼠后肢模型中动脉生成过程中白细胞募集

通过活体显微镜观察肠系膜缺血再灌注损伤后肠系膜小静脉中中性粒细胞细胞外陷阱的可视化

通过活体显微镜观察肠系膜缺血再灌注损伤后肠系膜小静脉中中性粒细胞细胞外陷阱的可视化