这项研究是支持的，部分的OIS维多利亚州政府的计划，全国卫生和医学研究理事会澳大利亚计划和项目赠款（JPF千石除尘）和研究生奖学金（四米歇尔）。

1。隔离颈动脉<ol><li> Euthanase10周龄SD大鼠，通过CO 2 / O 2的窒息。 </li><li>海关左，右颈总动脉与主动脉和心脏，确保最小伸展的船只。 </li><li>在冰冷的克雷布斯缓冲区单独的颈动脉从主动脉和心脏，并进行密切的夹层。 </li><li>在安装前，置于冰上克雷布斯隔离的船只。 </li></ol>约。时间= 45分钟 2。促发血管腔<ol><li>近端和远端血管腔冲洗克雷布斯注入carbogen天然气（95％O 2 5％的CO 2）保持在生理pH值的缓冲连接器，通过缓冲在37 ° C。 </li><li>确保油管和套管完全刷新和对齐。 </li><li>通过P1（近端）和P2（远端）传感器冲洗Kreb缓冲区。关闭水龙头，以确保在传感器无气泡。 </li><li>传感器连接到相应的连接器，并再次刷新更多克雷布斯缓冲区，确保无气泡。关上水龙头会议厅。 </li></ol>约。时间= 15分钟 3。加压容器室<ol><li>打开压力设备：伺服压力，压力显示器，蠕动泵（图1）。 </li><li>通过油管在20毫米汞柱（拨打1）确保无气泡，随着压力和压力伺服自动运行克雷布斯缓冲区的蠕动泵。 </li><li>连接管封闭P2传感器。压力将趋于稳定。 </li><li> P2的水龙头打开血管腔冲洗掉任何气泡，然后关闭掉。 </li><li>填写浴用Krebs缓冲液（5 - 7毫升）。 </li></ol>约。时间= 10分钟 4。安装船只<ol><li>在解剖显微镜放到每个套管黑色聚酯关系（图2）。 </li><li>移动套管和套管持有人除了安装¼船只上的P1导管（主动脉弓完）。确保不撕裂船只。 </li><li>使用填补了注射器用Krebs缓冲液轻轻冲洗多余的血液船只通过P1的传感器。关上的P1室。 </li><li>聚酯领带的小套管的安全船只。 </li><li>移动到P2套管安装套管/套管持有人接近。 </li><li>山末端的船只（长度¼）到远端插管。安全与聚酯领带。 </li></ol>约。时间= 20分钟 5。加压容器<ol><li>调整套管持有人，以确保没有弯曲或伸展在船只。 </li><li> P1和P2封闭的腔压力伺服自动切换到手动。压力伺服检查有没有在10秒内的压力不断减少。如果有，发生泄漏的连接和传感器需要的地方担保。 </li><li>调整压力伺服自动然后打开P2室。在显微镜下观察血管扩张时打开水龙头室。 </li><li>开关压力伺服手册。再次检查在10秒内的压力不断减少。如果有泄漏，则系统不施加压力，紧张，有可能是在船上的洞。 </li><li>切换回自动打开P1的会议厅。说明书上设置检查，压力保持稳定。 </li><li>如果没有泄漏，手动设置增加拨号2（40毫米汞柱）。 </li><li>调整自动，并在显微镜下观察的船只。如果有必要，调整的导管支架，以确保在vessel.Check没有手动设置泄漏的弯曲。 </li><li>增加表盘3（60毫米汞柱），4（80毫米汞柱），直到达到所需的压力是等重复步骤5.6 - 5.7。 </li></ol>约。时间= 15 - 30分钟 6。孵化的压力容器<ol><li>连接温度控制器设定到37 ° C。 </li><li>随着第二个蠕动泵灌注克雷布斯缓冲液（1毫升/分钟）入血管腔洗澡。 </li><li>宇航员室确保只有顶层洗澡的连接被删除。 </li><li>在37 ° C孵育1小时设置为自动压力压力容器。 </li><li>检查压力不泄漏（切换至手动）定期。 </li><li>通过简单地增加孵化过程中的化合物洗澡，可以观察各种药物干预的影响。 </li></ol>约。时间= 1小时 7。全血灌流压力容器<ol><li>孵化过程中获得至少7.5毫升全血/肝素40 U / mL的血液收集容器。在50毫升猎鹰在37 ° C。 </li><li>结束前10分钟的T他VybrantDil 10分钟（1:1000）孵育标签血液在37 °在C黑暗。 </li><li> 10分钟后，血液收集在清除任何气泡的注射器和附加到一个注射器泵热夹克在37 ° C。 </li><li> P1的传感器关闭室/船只和连接注射器和废物管。 </li><li>清除血液在1000μL/分钟通过废物管，以消除任何气泡。 </li><li>打开P1的会议厅。压力伺服会自动进行调整，以保持所需的压力。 </li><li>灌注100μL/ min的血。 </li><li>使用荧光显微镜，再加上一台数码相机记录两个领域的灌注船只为15秒1，3，5，7.5和10分钟。 </li><li>邮政灌注内皮细胞的完整性和功能，可以评估通过药理技术，如肌动描记和粘附分子的表达，可以通过免疫组织化学的决心，进一步验证了炎症反应。 </li></ol>约。时间= 15分钟 8。代表性的成果： 一个压力室设置的示意图如图1所示。随着数码相机，加上荧光显微镜结果立即通过现场录像可以可视化。代表视频图像看到图3中的白细胞被认为是附着内皮如果他们仍然固定为10秒。录像上连续循环坚持细胞，可以算作一个平均每场。虽然都低（ 图3A）和高（ 图3B）的压力会造成一定的附着量显着增加白细胞粘附在更高的腔内压力是看到，这也表明定量（ 图 4 ）。 <img alt="图1" src="/files/ftp_upload/3221/3221fig1.jpg" /> 图1。加压体外血管腔示意图 ，一个空心的容器，连接到近端（P1）的传感器和一个远端（P2）的传感器，使血压内的船舶操纵。灌注通过P1传感器和压力是保持通过P2传感器。 <img alt="图2" src="/files/ftp_upload/3221/3221fig2.jpg" /> 图2。黑色聚酯关系与联系Cannuala。连接到每个套管。 <img alt="图3" src="/files/ftp_upload/3221/3221fig3.jpg" /> 图3。代表动态的视频图像。细胞粘附在荧光（红色箭头），在80毫米汞柱（A）和120毫米汞柱（b）在灌注10分钟。 <img alt="图4" src="/files/ftp_upload/3221/3221fig4.jpg" /> 图4。斯普拉格Dawley白细胞粘附颈动脉后1小时在低潜伏期（80毫米汞柱）和高压（120毫米汞柱）。 *** P &lt;0.001，为2路反复的措施，使用Bonferroni后特别测试ANOVA分析。

<ol>
	<li>Lopez, A. D., Mathers, C. D., Ezzati, M., Jamison, D. T., Murray, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+and+regional+burden+of+disease+and+risk+factors.">Global and regional burden of disease and risk factors.</a> Lancet. 367, 1747-1757 (2001).</li><li>Szczech, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+kidney+injury+and+cardiovascular+outcomes+in+acute+severe+hypertension.">Acute kidney injury and cardiovascular outcomes in acute severe hypertension.</a> Circulation. 121, 2183-2191 .</li><li>Rodriguez-Garcia, J. L., Botia, E., de La Sierra, A., Villanueva, M. A., Gonzalez-Spinola, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Significance+of+elevated+blood+pressure+and+its+management+on+the+short-term+outcome+of+patients+with+acute+ischemic+stroke.">Significance of elevated blood pressure and its management on the short-term outcome of patients with acute ischemic stroke.</a> Am J Hypertens. 18, 379-384 (2005).</li><li>Levy, D., Larson, M. G., Vasan, R. S., Kannel, W. B., Ho, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+progression+from+hypertension+to+congestive+heart+failure.">The progression from hypertension to congestive heart failure.</a> JAMA. 275, 1557-1562 (1996).</li><li>Ley, K., Laudanna, C., Cybulsky, M. I., Nourshargh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+to+the+site+of+inflammation:+the+leukocyte+adhesion+cascade+updated.">Getting to the site of inflammation: the leukocyte adhesion cascade updated.</a> Nat Rev Immunol. 7, 678-689 (2007).</li><li>Riou, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+pressure+promotes+monocyte+adhesion+to+the+vascular+wall.">High pressure promotes monocyte adhesion to the vascular wall.</a> Circ Res. 100, 1226-1233 (2007).</li><li>Ley, K., Gaehtgens, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial,+not+hemodynamic,+differences+are+responsible+for+preferential+leukocyte+rolling+in+rat+mesenteric+venules.+Circ+Res.">Endothelial, not hemodynamic, differences are responsible for preferential leukocyte rolling in rat mesenteric venules. Circ Res.</a> 69, 1034-1041 (1991).</li><li>Bernhagen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MIF+is+a+noncognate+ligand+of+CXC+chemokine+receptors+in+inflammatory+and+atherogenic+cell+recruitment.">MIF is a noncognate ligand of CXC chemokine receptors in inflammatory and atherogenic cell recruitment.</a> Nat Med. 13, 587-596 (2007).</li><li>Woollard, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiological+levels+of+soluble+P-selectin+mediate+adhesion+of+leukocytes+to+the+endothelium+through+Mac-1+activation.">Pathophysiological levels of soluble P-selectin mediate adhesion of leukocytes to the endothelium through Mac-1 activation.</a> Circ Res. 103, 1128-1138 (2008).</li><li>Eberth, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+pulsatility+in+hypertensive+carotid+artery+growth+and+remodeling.">Importance of pulsatility in hypertensive carotid artery growth and remodeling.</a> J Hypertens. 27, 2010-2021 (2009).</li><li>Cooke, J. P., Rossitch, E., Andon, N. A., Loscalzo, J., Dzau, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+activates+an+endothelial+potassium+channel+to+release+an+endogenous+nitrovasodilator.">Flow activates an endothelial potassium channel to release an endogenous nitrovasodilator.</a> J Clin Invest. 88, 1663-1671 (1991).</li></ol>

这项研究的协议是由阿尔弗雷德医学研究和教育专用区动物伦理委员会和阿尔弗雷德医院伦理委员会的批准。

这是修改后的的方法来研究实时加压条件下的完整的隔离血管内皮白细胞粘附。单独的血管腔灌注，使一个大小鼠和大鼠血管的促炎症株的快速验证。启用的压力操纵允许动态的细胞间的相互作用，以观察从低到非常高的腔内压力，从而更好地模拟生理和病理条件。血管直径也可以用足够的细胞成像方案，因此剪切流动和速率可确定，因此操作。 凭借其肌动描记能力，放置在洗澡的药理干预添加另一个层面可能的实验条件下，这种模式使机理和信号通路的研究。虽然内皮保护不能在压力操纵证实，对乙酰胆碱和PE的反应，可进行后灌注 9 。 应该指出的是，此设置演示腔内压力的影响，在细胞与细胞之间的相互作用不是搏动性血流量，也不收缩压或舒张压的影响。此外，白细胞粘附的急性压力的变化进行观察时，这种设置可以还利用来看待慢性压力的影响（即增加孵育时间和使用一种慢性压力的动物模型）。斯普拉格Dawley颈总动脉中演示了这一套，但其他菌株和物种可能用于套管大小适当调整。事实上，重要的是要注意动物的年龄和体重的影响船只的大小，因此，设立需要因材施教，为每艘船舶。关闭并仔细解剖结缔组织可以可视化极大地提高白细胞。

<table border="1"><tbody><tr><th>试剂/设备名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td>显微镜</td><td>卡尔蔡司公司</td><td>立体声发现五.20 </td><td></td></tr><tr><td>博士2000注射泵</td><td>哈佛器械</td><td> 70-2016 </td><td></td></tr><tr><td>数码相机和控制器</td><td>滨松ORCA的二</td><td> C4742 - 95 </td><td></td></tr><tr><td>荧光照明系统</td><td>流明动态</td><td>的X - Cite 120 </td><td></td></tr><tr><td>货船商会</td><td>生活系统设备</td><td> CH/1/SH </td><td></td></tr><tr><td>压力伺服控制器和蠕动泵</td><td>生活系统设备</td><td> PS - 200 </td><td></td></tr><tr><td>灌注血压计</td><td>生活系统设备</td><td> AM - 4 </td><td></td></tr><tr><td> 2个压力传感器</td><td>生活系统设备</td><td> PT - F </td><td></td></tr><tr><td>温度控制器</td><td>生活系统设备</td><td> TC - 01 </td><td></td></tr><tr><td>蠕动泵</td><td> Instech实验室，公司</td><td> P720 </td><td></td></tr><tr><td> VybrantDil细胞标签解决方案</td><td> Invitrogen公司</td><td> V - 22885 </td><td>使用1:1000 </td></tr></tbody></table>

imaging leukocyte adhesion to the vascular endothelium at high intraluminal pressure

据报道，约四分之一的人口在世界范围内，高血压和死亡率全球领先的生物医药风险因素。在血管性高血压与血管内皮功能障碍和增加炎症导致动脉粥样硬化和各种疾病，如慢性肾脏病 ，中风3和心力衰竭4国。在血管炎症导致动脉粥样硬化的最初步骤，其中包括通过血管内皮细胞的滚动，圈养，坚持和随后轮回白细胞的粘附级联。招聘和血管内皮细胞白细胞的积累是介导的粘附分子如血管细胞粘附分子-1（VCAM - 1），细胞内的细胞粘附分子1（ICAM - 1）和E -选择以及增加的上调细胞因子和趋化因子的释放和活性氧上调5。，帮助在体外培养的方法，如静态粘附检测，以确定参与细胞-细胞粘附机制以及细胞粘附分子的分析。在以前聘用的方法在体外研究表明，急性血管内皮细胞上的压力增加可导致单核细胞粘附，粘附分子和炎症标志物的上调6然而，在许多体外试验类似，这些研究结果并没有被实时执行生理流动条件下，也与全血。因此， 在体内实验中越来越多地利用在动物模型中表现出血管炎症和斑块的发展。活体显微镜是目前广泛使用的评估白细胞粘附，轧件，迁移和轮回7-9。当上的白细胞内皮细胞粘附在体内研究相结合的压力的影响不太广泛。这样的一个研究探讨动脉生长和重塑，但炎症标志物的流动和剪切的实时效果，只有通过免疫组织化学10评估。在这里，我们提出了一个完好加压的血液，使用全血灌注血管的实时记录白细胞粘附的模型。方法是体外血管腔灌注模型9，使实时分析的白细胞与内皮完整的血管粘合剂相互作用的修改。我们的修改，使操纵的腔内压力高达200毫米汞柱，不仅在生理流动条件，但也压力条件下的研究。虽然压力myography系统此前已证明，观察血管壁和管腔直径11以及血管收缩，这是首次展示实时的白细胞-内皮相互作用。在这里，我们展示了收获大鼠颈总动脉插管至一个定制的流室加上荧光显微镜技术，使用。船只室配有一个大的底部，让一个大口径物镜工作距离短，以形象船只玻璃罩。此外，选定的激动剂和/或拮抗剂可以利用控制细胞粘附的机制，以进一步调查。活体显微镜这种方法的优点包括没有介入微创手术，因此可以得到更高的吞吐量。这种方法也可以使用本地化抑制剂治疗所需的船只，而活体不仅能使全身抑制剂治疗。

Watch this Scientific Journal Video about Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure at JoVE.com

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

这是一个可视化白细胞粘附到内皮收获压力容器的方法。该技术使研究不同的腔内压力高达200毫米汞柱，从而模拟高血压的病理生理条件下剪切流动的血管粘连。

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. ...

imaging-leukocyte-adhesion-to-vascular-endothelium-at-high

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JoVE Journal

Immunology and Infection

15.5K 次观看.  Monash University. 这是一个可视化白细胞粘附到内皮收获压力容器的方法。该技术使研究不同的腔内压力高达200毫米汞柱，从而模拟高血压的病理生理条件下剪切流动的血管粘连。

视频: Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

白细胞的归位和迁移的非侵入性的成像在体内</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

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免疫与感染

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

评估气孔反应活细菌细胞，采用全叶成像

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

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 ...

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

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

一个流程粘附实验来研究白细胞招聘剪切应力的人力肝窦内皮细胞条件下

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

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

人中性粒细胞流动室粘附实验

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

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

可扩展的高通量选择从噬菌体展示合成抗体库

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

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

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

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

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

在体外和体内模型来研究细菌粘附于血管壁下流动条件

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

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 ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

在剪切粘结力的测定T淋巴细胞粘附分子相互作用的研究

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...