这项工作是支持的，部分由美国国立卫生研究院（：P30 HL101302和RO1 HL109439 JC）和美国心脏协会（SDG 5270005 JC）的补助金。答：Barazia是支持的，由一个T32HL007829美国国立卫生研究院培训补助金。

1。活体显微镜（图1A）的制备<ol><li>准备灌流缓冲液（125 mM氯化钠，4.5 mM KCl中，2.5mM的CaCl 2的 ，1毫摩尔的MgCl 2，和17毫摩尔的NaHCO 3，pH为7.4）。 </li><li>打开上的循环水浴中，在37℃的缓冲液，以保持温度和热控制毯用氮气曝气缓冲液（5％CO 2的平衡用氮气）。 </li><li>打开显微镜系统（萨特LAMBDA DG-4高速波长变换器，BX61W工作站计算机上，奥林巴斯显微镜，MPC-200多机械手，净资产收益率200台控制器，高速相机，并增强）。 </li><li>对于血管炎症模型，使用100％的分色镜。若要通过激光损伤诱导血栓形成，代替100％的50/50％的反射镜的反射镜，并打开一个微点激光消融系统。 </li></ol> 2。准备提睾肌活体显微镜（图1B） <ol><li>麻醉一腹腔注射氯胺酮（125毫克/千克体重（BW））和甲苯噻嗪（12.5 mg / kg体重）的雄性小鼠（6〜8周龄C57BL / 6）。大学伊利诺伊实验动物护理和使用委员会批准所有的动物保健和实验程序。对于一个血管炎症模型中，注入鼠TNF-α（0.5微克在250μl生理盐水）intrascrotally成鼠标3小时之前成像（2-2.5小时，在手术前）。 </li><li>鼠标放置在37℃下的热控制的毛毯上的活体显微镜托盘。 </li><li>拉了皮肤表面上的脖子上使用产钳，切割水平中线的颈部皮肤1厘米的剪刀。 </li><li>轻轻地打开颈部肌肉，用钝头剪刀，取出气管周围的肌肉。 </li><li>一个PE90管插入气管插管，以消除呼吸困难。 </li><li>轻轻地取出颈肌的左侧，并从周围组织中分离出颈静脉。 </li><li> Ç每年迟到PE10管插入左颈静脉输液的抗体和额外麻醉药。 </li><li>轻轻拉出，水平切开阴囊皮肤。整个实验过程中为了维持组织完整性，预热的缓冲液灌流超过手术字段。 </li><li>按于下腹部有一个镊子，并与其他镊子，小心地拉出一个睾丸。 </li><li>删除周围的睾丸周围的结缔组织。 </li><li>垂直切开提睾肌和扁平的肌肉，通过固定的外围活体显微镜盘的一个盖玻片上。 </li><li>允许的肌肉，以稳定数据采集前的10〜20分钟。我们的研究中被选择用于提睾肌动脉（血栓形成）和静脉（血管炎）的直径为30-45微米。 </li><li>为了维持麻醉的条件下，注入相同的麻醉剂，大约每30分钟通过颈静脉cannulus 30-50毫升。 </li></ol> &lt;P类=“j​​ove_title”&gt; 3。 TNF-α诱导的血管炎症的活体显微镜<ol><li>安置后的活体显微镜，打开SlideBook 5.0软件的鼠标，点击菜单栏上的“焦点访谈”窗口中设置相应的光学和目标（60X）。 </li><li>输注Dylight 488-共轭的大鼠抗小鼠CD42c（0.1微克/克体重在100μl生理盐水）和Alexa Fluor 647 - 共轭的大鼠抗小鼠的Gr-1抗体（0.05微克/克体重在100μl生理盐水）通过颈cannulus中。 </li><li>进入菜单栏，点击在工具栏上的“图像捕捉”。 </li><li>会弹出一个新的菜单，在“图像”，选择“斌因子”的景点，并选择“640”宽度“460”的高度。 </li><li>在“过滤设置”，选中复选框的渠道，你想揭露和设置曝光时间为每个。例如，打开通道：FITC通道10毫秒，50毫秒，和Cy5通道：50毫秒。 </li><li>选择“时间推移捕捉”调整的时间点和时间间隔的数量。佛山r的炎症模型，200毫秒的时间间隔（经历的总时间：5分钟）的时间点的数目被设置为1,000-1,500。 </li><li>点击“Test”在指定的曝光时间，该频道看到一个单一的平面图像。调整曝光时间和/或显微镜的光，直到直方图显示了足够的强度分布（对于多通道采集，重复此过程每个通道）。 </li><li>一旦所有的参数设置（通道，曝光时间，等等）中的“图像捕捉”，选择“开始”开始捕捉。 </li><li>监控发炎提睾小静脉的血管炎症模型的上半部分中的5分钟的轧制和粘附的血小板和嗜中性粒细胞。八至十个不同的静脉都记录在一个鼠标。 </li><li>画像被使用的总放大倍数为60X（1.0 NA水浸物镜）给窗口大小为157微米×118微米。 </li><li>实验完成后，将小鼠安乐死经由宫颈癌dislocatioN。 </li></ol> 4。小静脉炎症模型的数据分析<ol><li>打开SlideBook 5.0软件，点击菜单栏上的“文件夹”，打开一个文件。 </li><li>点击下拉通道菜单，并选择“打开”，FITC，Cy5的，等等。“ </li><li>点击“归一化（红色和绿色的条）”，并选择通道。拖动的左侧或右侧的红色和绿色的酒吧大致优化的荧光信号。 </li><li>单击“应用”和“确定”以更新的形象。 </li><li>单击“缩略图”按钮，选择当前显示为默认值。 </li><li>播放拍摄的间隔拍摄的图像，滚动和粘附的中性粒细胞计数在5分钟内。 </li><li> 分析血小板血栓形成 ，到“面具”，然后选择“创建”。 </li><li>命名口罩，并注明“在目前的图像”。 </li><li>按“大”，“选框工具”在主视图中的铅笔图标。 </li><li>颜色区域外的船只在主视图中设置背景遮罩。 </li><li>转到吨“O”面具“下，单击”复制这架飞​​机“，单击”复制面具在目前的时间点“，并选择”所有时间点“，然后单击”OK“。 </li><li> 要计算背景信号中 ，“统计”，单击“”面膜统计“。 </li><li>单击“目前的2D时间的推移或四维图像（S）”，在图像范围，然后选择“整个面具”戴面具的范围。在功能方面，选择“运行时间（HH：MM：毫秒）”根据拍摄日期，选择“根据形态测量的面积（像素）”，选择“最大强度”下的强度，并单击“导出”（此统计方法，使我们能够计算自体荧光强度（背景信号））。 </li><li>在MS Office Excel中打开文本文件和记录期间的背景信号计算出的平均值。在这样的背景荧​​光强度。 </li><li> 要确定血栓的强度 ，点击“大铅笔图标”再次在“选框工具”在主视图中。 </li><li>在主视图中设置血小板信号的容器内的颜色。 </li><li> “面具”，单击“复制这架飞​​机”，单击“复制面具在目前的时间点”，选择“所有时间点”，并单击“确定”。 </li><li> “统计”，单击“”面膜统计“。 </li><li>单击“目前的2D时间的推移或四维图像（S）”，在图像范围，然后选择“整个面具”戴面具的范围。在功能方面，选择“运行时间（HH：MM：毫秒）”根据拍摄日期，选择“面积（像素）”根据形态测量学中，选择“求和强度”下的强度，并单击“导出”（此统计方法使我们能够计算在所述容器内的荧光强度）。 </li><li>打开MS Office Excel中的文本文件中。的总和在容器内部的强度减去背景强度x面积（像素）的平均值，计算出荧光信号的血小板。这是血小板血栓的荧光强度。 </li><li>重复此步骤3-5个不同的小鼠在30个不同的静脉。然后，计算出的荧光强度中位值的血小板。 </li><li> 对分析，滚滚涌入的中性粒细胞，中性粒细胞的滚动和粘附确定超过5分钟，在每个小静脉，表现为细胞数量每分钟。数固定为30秒，慢慢地爬（&lt;10微米超过30秒），但没有翻身的粘附中性粒细胞的数量，表现为细胞数量每5分钟。 </li></ol> 5。激光诱导小动脉血栓形成的活体显微镜 5-1消融激光校准<ol><li>将镜像幻灯片在显微镜阶段，应用到幻灯片顶部的一小滴蒸馏水，调整的力度和重点使用的目镜。 </li><li>打开“焦点访谈”窗口“，然后选择”选项卡中SlideBook 5.0软件的“FRAP”的。 </li><li>设置激光功率在40-50，设定两个“双击重复”和“双击大小”8。标记的“指南”框中，带来了一组十字线。 </li><li>选择“箭头”图标从任务栏Øn的屏幕顶部。 </li><li>在“FRAP对齐”选项卡中，单击“下火”。应该出现在显示器左下角的一个小点。一旦在现场出现，双击它的中心。按下按钮后，另一点应出现上述第一点的权利，双击它。重复此为16点（16 个点会出现在右下角。），点击“保存”完成后，更新校准参数。 </li><li>要测试的校准的精度，单击“上的”中心“按钮的小光点应出现在屏幕的中心。如果没有，请重新校准，直到达到所需的精度。 </li></ol> 5-2激光诱导小动脉血栓形成<ol><li>在显微镜下阶段将麻醉小鼠（2-2至2-12的程序）。 </li><li>在“捕获”窗口中，选择一个协议或创建一个新的。设置如下：CY5（70毫秒暴露），公开组（10 MSEC曝光）和FITC（50毫秒暴露）。图像被捕获超过20分钟（约2,400时间点的间隔时间为500毫秒）。 </li><li>输注Dylight 488共轭抗小鼠CD42c和Alexa Fluor 647-共轭抗小鼠的Gr-1抗体如上所述。 </li><li>优化显微镜参数包括荧光强度和亮场图像所描述的3-3到3-7。 </li><li>点击“测试”按钮，以确保适当的明场或抗体信号强度，小动脉的位置和重点。 </li><li>单击“开始”，启动图像采集过程中。 </li><li>消融激光发射前，请务必将鼠标指针图标已被选中，血管壁明确的重点。 </li><li>要发射的激光，双击一个点2-3微米的血管壁内部。小动脉内皮细胞的损伤会导致视觉形状的变化，应该是明显的亮场图像，其次是血小板聚集。监视器血栓5分钟后，激光损伤的形成。 </li><li>五分钟后，激光损伤（现在应该是稳定的血栓的大小）暂停和取消捕获。接着，启动捕获与2400的时间点，与一个500毫秒的时间间隔为20分钟记录粘附的血小板和滚动/粘附中性粒细胞。三至五年的动脉被记录在一个鼠标。 </li><li>实验完成后，通过颈椎脱臼处死小鼠。 </li></ol> 6。小动脉血栓形成模型的数据分析。 <ol><li>以上步骤4.1至4.5。 </li><li> 为了获得背景荧光强度血小板血栓的形成过程中 ，以上步骤4.7到4.14。 </li><li> “面具”，单击“分类”，选择FITC通道中，并插入背景信号的平均值低。点击“Apply”和“OK”。 </li><li> 为了分析血小板血栓的荧光强度（Dylight 488-共轭的抗CD42c抗体）， </st荣&gt;以上步骤4.18至4.19。要计算的抗体信号，“萨姆强度”中减去背景信号（“最大强度”x“的面积（像素数）”）。这是血小板血栓的荧光强度。 </li><li> 为了量化滚动和粘附中性粒细胞的 ，玩时光倒流，清晰可见，滚动超过20分钟的血小板血栓的细胞数一数。轧制相互作用定义为嗜中性粒细胞的速度减少，而与至少2秒的血小板血栓。遗民中性粒细胞被定义为任何仍然附着的血小板血栓的嗜中性粒细胞，至少2分钟。表现为细胞数量每20分钟滚动和粘附中性粒细胞的数量。轧制速度计算利用粒子跟踪系统。 </li></ol>

<ol>
	<li>Wagner, D. D., Frenette, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vessel+wall+and+its+interactions.">The vessel wall and its interactions.</a> Blood. 111, 5271-5281 (2008).</li><li>Nieswandt, B., Kleinschnitz, C., Stoll, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischaemic+stroke:+a+thrombo-inflammatory+disease.">Ischaemic stroke: a thrombo-inflammatory disease.</a> J. Physiol. 589, 4115-4123 (2011).</li><li>Yang, J., Furie, B. C., Furie, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+P-selectin+glycoprotein+ligand-1:+its+role+as+a+selectin+counterreceptor+in+leukocyte-endothelial+and+leukocyte-platelet+interaction.">The biology of P-selectin glycoprotein ligand-1: its role as a selectin counterreceptor in leukocyte-endothelial and leukocyte-platelet interaction.</a> Thromb. Haemost. 81, 1-7 (1999).</li><li>Zarbock, A., Polanowska-Grabowska, R. K., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-neutrophil-interactions:+linking+hemostasis+and+inflammation.">Platelet-neutrophil-interactions: linking hemostasis and inflammation.</a> Blood Rev. 21, 99-111 (2007).</li><li>Chen, J., Lopez, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+platelets+with+subendothelium+and+endothelium.">Interactions of platelets with subendothelium and endothelium.</a> Microcirculation. 12, 235-246 (2005).</li><li>Konstantopoulos, K., 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=Venous+levels+of+shear+support+neutrophil-platelet+adhesion+and+neutrophil+aggregation+in+blood+via+P-selectin+and+beta2-integrin.">Venous levels of shear support neutrophil-platelet adhesion and neutrophil aggregation in blood via P-selectin and beta2-integrin.</a> Circulation. 98, 873-882 (1998).</li><li>Maugeri, N., de Gaetano, G., Barbanti, M., Donati, M. B., Cerletti, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+platelet-polymorphonuclear+leukocyte+interactions:+new+clues+to+the+antithrombotic+properties+of+parnaparin,+a+low+molecular+weight+heparin.">Prevention of platelet-polymorphonuclear leukocyte interactions: new clues to the antithrombotic properties of parnaparin, a low molecular weight heparin.</a> Haematologica. 90, 833-839 (2005).</li><li>Hidalgo, A., 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=Heterotypic+interactions+enabled+by+polarized+neutrophil+microdomains+mediate+thromboinflammatory+injury.">Heterotypic interactions enabled by polarized neutrophil microdomains mediate thromboinflammatory injury.</a> Nat. Med. 15, 384-391 (2009).</li><li>Cho, J., Furie, B. C., Coughlin, S. R., Furie, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+role+for+extracellular+protein+disulfide+isomerase+during+thrombus+formation+in+mice.">A critical role for extracellular protein disulfide isomerase during thrombus formation in mice.</a> J. Clin. Invest. 118, 1123-1131 (2008).</li><li>Cho, J., 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=Protein+disulfide+isomerase+capture+during+thrombus+formation+in+vivo+depends+on+the+presence+of+beta3+integrins.">Protein disulfide isomerase capture during thrombus formation in vivo depends on the presence of beta3 integrins.</a> Blood. 120, 647-655 (2012).</li><li>Gross, P. L., Furie, B. C., Merrill-Skoloff, G., Chou, J., Furie, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte-versus+microparticle-mediated+tissue+factor+transfer+during+arteriolar+thrombus+development.">Leukocyte-versus microparticle-mediated tissue factor transfer during arteriolar thrombus development.</a> Journal of Leukocyte Biology. 78, 1318-1326 (2005).</li><li>Falati, S., Gross, P., Merrill-Skoloff, G., Furie, B. C., Furie, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+in+vivo+imaging+of+platelets,+tissue+factor+and+fibrin+during+arterial+thrombus+formation+in+the+mouse.">Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse.</a> Nat. Med. 8, 1175-1181 (2002).</li><li>Barthel, S. R., 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=Alpha+1,3+fucosyltransferases+are+master+regulators+of+prostate+cancer+cell+trafficking.">Alpha 1,3 fucosyltransferases are master regulators of prostate cancer cell trafficking.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 19491-19496 (2009).</li><li>Trzpis, M., McLaughlin, P. M., de Leij, L. M., Harmsen, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+cell+adhesion+molecule:+more+than+a+carcinoma+marker+and+adhesion+molecule.">Epithelial cell adhesion molecule: more than a carcinoma marker and adhesion molecule.</a> The American Journal of Pathology. 171, 386-395 (2007).</li><li>Junt, T., 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=Dynamic+visualization+of+thrombopoiesis+within+bone+marrow.">Dynamic visualization of thrombopoiesis within bone marrow.</a> Science. 317, 1767-1770 (2007).</li><li>Egan, C. E., Sukhumavasi, W., Bierly, A. L., Denkers, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+multiple+functions+of+Gr-1(+)+cell+subpopulations+during+microbial+infection.">Understanding the multiple functions of Gr-1(+) cell subpopulations during microbial infection.</a> Immunologic Research. 40, 35-48 (2008).</li></ol>

作者宣称没有竞争的财务权益。

在这里，我们描述了一个详细的协议，实时荧光活体显微镜可视化激活的内皮细胞在血管炎症和血栓形成的异型血小板中性粒细胞的相互作用。在此之前，类似的荧光显微镜方法研究血栓形成和血管炎症的分子机制。8,12由于异型细胞与细胞间的相互作用可能是重要的，在损伤部位的血管闭塞，这种技术将是一个有价值的工具血管疾病的细胞和分子机制的研究。实时成像技术的优点是监测?...

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>7647-14-5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>7447-40-7</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CaCl2 2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>10035-04-8</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgCl2 6H2O</td>
			<td>Fisher Scientific</td>
			<td>7791-18-6</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Fisher Scientific</td>
			<td>144-55-8</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.9% NaCl Saline</td>
			<td>Hospira</td>
			<td>0409-4888-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Hospira</td>
			<td>0409-2051-05</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Lloyd</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Intramedic Tubing (PE 90)</td>
			<td>BD Diagnostics</td>
			<td>427421</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Intramedic Tubing (PE 10)</td>
			<td>BD Diagnostics</td>
			<td>427401</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Murine TNF-&alpha;</td>
			<td>R&amp;D Systems</td>
			<td>410-MT</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dylight 488- labeled rat anti-mouse CD42b antibody</td>
			<td>Emfret Analytics</td>
			<td>X488</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647-conjugated anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody</td>
			<td>BioLegend</td>
			<td>108418</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NESLAB EX water bath/circulator</td>
			<td>Thermo-Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Olympus BX61W microscope</td>
			<td>Olympus</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TH4-100 Power</td>
			<td>Olympus</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lambda DG-4</td>
			<td>Sutter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MPC-200 multi-manipulator</td>
			<td>Sutter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ROE-200 stage controller</td>
			<td>Sutter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>C9300 high-speed camera</td>
			<td>Hamamatsu</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Intensifier</td>
			<td>Video Scope International</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ablation Laser</td>
			<td>Photonic Instruments, Inc.</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SlideBook 5.0</td>
			<td>Intelligent Imaging Innovations</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

real-time imaging of heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice

相互作用的活化的血小板和白细胞（主要是嗜中性粒细胞），活化的内皮细胞介导的血栓形成和血管炎症。1,2期间在现场小动脉损伤的血栓形成，血小板附着的激活的内皮细胞和内皮下基质蛋白支持中性粒细胞的轧制和粘附性。3相反，小静脉的炎症条件下，嗜中性粒细胞激活的内皮细胞的粘附，可以支持循环血小板的附着和蓄积。异型血小板中性粒细胞聚集需要由特定的受体计数器受体细胞之间的相互作用的顺序过程4，它是已知的，活化的内皮细胞释放粘附分子如von Willebrand因子，由此开始在高剪切条件下，血小板的附着和蓄积。5另外，支持中性粒细胞激活内皮细胞表达选择素的滚动和粘附ð细胞间粘附分子-1（ICAM-1），分别在低剪切条件下，4血小板P-选择交互与嗜中性粒细胞通过P-选择素糖蛋白配体-1（PSGL-1），从而诱导活化的嗜中性粒细胞β2整合和坚定两种类型的细胞之间的粘附。尽管在体外实验中，全血或分离的细胞，6,7异型血小板，中性粒细胞的相互作用确定的进步，这些研究不能操纵在血管疾病的氧化应激条件。在这份报告中，用鼠标血小板和中性粒细胞的标记荧光标记的特异性抗体，我们描述了一个详细的活体显微协议来监控异型相互作用的血小板和中性粒细胞活化的内皮细胞在TNF-α诱导的炎症或激光诱导提睾肌活体小鼠微血管的损伤。

使用详细的活体显微镜分析，异型血小板中性粒细胞上的激活的内皮细胞的相互作用是可视化的荧光标记的血小板（CD42c）或嗜中性粒细胞的标记（的Gr-1）到活体小鼠抗输注。 在一个模型中的TNF-α诱导的小静脉的炎症，最轧制的中性粒细胞稳定地粘附到内皮细胞想必与ICAM-1的相互作用激活β2整合在记录期间（3-4.5小时后注射的TNF-α， 图2A 8滚动轴承和粘附...

Watch this Scientific Journal Video about Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mic

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

在这里，我们报告荧光活体显微镜可视化激活的内皮细胞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相互作用的实验技术。显微技术将是很有价值的研究血管疾病的分子机制和病理生理条件下测试药物制剂。

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 ...

real-time-imaging-heterotypic-platelet-neutrophil-interactions-on

Research

JoVE Journal

Immunology and Infection

15.6K 次观看.  University of Illinois at Chicago. 在这里，我们报告荧光活体显微镜可视化激活的内皮细胞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相互作用的实验技术。显微技术将是很有价值的研究血管疾病的分子机制和病理生理条件下测试药物制剂。

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

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of &beta;-arrestins3. &beta;-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization 4-6.
Leukotriene B4 (LTB4) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB4 receptor 1 (BLT1; a high affinity receptor) and LTB4 receptor 2 (BLT2; a low affinity receptor)7-9. The LTB4-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis10-17. The current paper describes the methodologies developed to monitor LTB4-induced leukocyte migration and the interactions of BLT1 with &beta;-arrestin and , receptor translocation in live cells using microscopy imaging techniques18-19.
Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described 20-21. These cells were tested in live cell imaging methods to demonstrate LTB4 induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and &beta;-arrestin1 tagged with green fluorescent protein (&beta;-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &amp;#946;-arrestin

This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.

白三烯B实时成像 4介导的细胞迁移和BLT1与β- arrestin的相互作用

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

科研

免疫与感染

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. In the vasculature hypertension is associated with endothelial dysfunction and increased inflammation leading to atherosclerosis and various disease states such as chronic kidney disease2, stroke3 and heart failure4. An initial step in vascular inflammation leading to atherogenesis is the adhesion cascade which involves the rolling, tethering, adherence and subsequent transmigration of leukocytes through the endothelium. Recruitment and accumulation of leukocytes to the endothelium is mediated by an upregulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin as well as increases in cytokine and chemokine release and an upregulation of reactive oxygen species5. In vitro methods such as static adhesion assays help to determine mechanisms involved in cell-to-cell adhesion as well as the analysis of cell adhesion molecules. Methods employed in previous in vitro studies have demonstrated that acute increases in pressure on the endothelium can lead to monocyte adhesion, an upregulation of adhesion molecules and inflammatory markers6 however, similar to many in vitro assays, these findings have not been performed in real time under physiological flow conditions, nor with whole blood. Therefore, in vivo assays are increasingly utilised in animal models to demonstrate vascular inflammation and plaque development. Intravital microscopy is now widely used to assess leukocyte adhesion, rolling, migration and transmigration7-9. When combining the effects of pressure on leukocyte to endothelial adhesion the in vivo studies are less extensive. One such study examines the real time effects of flow and shear on arterial growth and remodelling but inflammatory markers were only assessed via immunohistochemistry10. Here we present a model for recording leukocyte adhesion in real time in intact pressurised blood vessels using whole blood perfusion. The methodology is a modification of an ex vivo vessel chamber perfusion model9 which enables real-time analysis of leukocyte -endothelial adhesive interactions in intact vessels. Our modification enables the manipulation of the intraluminal pressure up to 200 mmHg allowing for study not only under physiological flow conditions but also pressure conditions. While pressure myography systems have been previously demonstrated to observe vessel wall and lumen diameter11 as well as vessel contraction this is the first time demonstrating leukocyte-endothelial interactions in real time. Here we demonstrate the technique using carotid arteries harvested from rats and cannulated to a custom-made flow chamber coupled to a fluorescent microscope. The vessel chamber is equipped with a large bottom coverglass allowing a large diameter objective lens with short working distance to image the vessel. Furthermore, selected agonist and/or antagonists can be utilized to further investigate the mechanisms controlling cell adhesion. Advantages of this method over intravital microscopy include no involvement of invasive surgery and therefore a higher throughput can be obtained. This method also enables the use of localised inhibitor treatment to the desired vessel whereas intravital only enables systemic inhibitor treatment.

This is a method to visualise leukocyte adhesion to the endothelium in harvested pressurised vessels. The technique enables studying vascular adhesion under shear flow with differing intraluminal pressures up to 200 mmHg thus mimic-ing the pathophysiological conditions of high blood pressure.

成像白细胞粘附到血管内皮细胞在高腔内压力

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. ...

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

小胶质细胞的小分子RNA检测在正常中枢神经系统的实时PCR技术在神经发炎

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

T细胞实时成像实时信号复合物的形成

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

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

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

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 ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.
We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNF&#945;) and found the presence of CD16+ cells to be significantly higher on TNF&#945; stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

一种新型<em&gt;在体外</em&gt;模型研究人类全血细胞间的相互作用

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions.&#160;The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model.&#160;In this respect, the present manuscript describes the use of zebrafish as an&#160;in vivo&#160;model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.

破译和成像发病机制与盘带<em&gt;脓肿分枝杆菌</em&gt;斑马鱼胚胎

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen ...

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 ...

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. 
In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay.
Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). 
The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage&#8217;s metabolic phenotype and inflammatory status.

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage&#8217;s metabolism and phenotype.

偏光M1和M2骨髓来源的巨噬细胞的使用实时外流量分析代谢表征

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network.
By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives&#160;valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules.
The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.

We present an in vitro vascular disease model to investigate whole blood interactions with patient-derived endothelium. This system allows the study of thrombogenic properties of primary endothelial cells under various circumstances. The method is especially suited to evaluate in situ thrombogenicity and anticoagulation therapy during different phases of coagulation.

实时研究全血-内皮相互作用和血栓动力学的体外微流体疾病模型

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The ...