Name: quantitation of endothelial cell adhesiveness in vitro
Uploaded: 2015-06-18T15:00:00.000+00:00
Duration: 10 min 24 s
Description: Watch this Scientific Journal Video about Quantitation of Endothelial Cell Adhesiveness In Vitro at JoVE.com

We are very grateful to Simon Bouffler for his full support and to Public Health England for infrastructure support. This work was supported by Public Health England throughthe National Institute for Health Research (NIHR) grant. This report is work commissioned by the NIHR. The views expressed in this publication are those of the authors and not necessarily those of the NHS, NIHR or the Department of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

1.准备玻璃盖玻片<ol><li>消毒直径12mm圆形盖玻片通过浸泡在70％乙醇中至少10分钟，偶尔搅拌，以确保所有的盖玻片的乙醇的总暴露。倒入玻璃盖玻片和乙醇进入无菌细胞培养皿（无论是6厘米直径10厘米）。 </li><li>有一对无菌细5B镊子，拾取和放置每个单独的玻璃盖玻片成24孔簇平板的孔。小心确保盖玻片不接触孔的侧面和大致在井的中间。 </li><li>离开未覆盖的24孔丛板盖玻片在流柜干燥。这大约需要10分钟</li><li>在此期间，由于Hank氏平衡盐溶液（HBSS），以0.1mg / ml的稀释10mg / ml的人纤连蛋白制备的涂料混合物。 </li><li>当盖玻片是干的，吸移管100微升的涂敷液涂敷到每个玻璃共verslip所以该溶液仅覆盖玻璃而不围绕着阱的外面汇集。将覆盖24孔簇板中的细胞培养孵化器的至少一个小时。 </li><li>在使用前，除去包衣液。在相同的涂层溶液可以转移到无菌管中并用高达三倍与疗效没有可见损失。 </li></ol> 2.制备内皮细胞单层<ol><li>培养的人冠状动脉内皮细胞（EC），在培养基中，在37℃和5％的二氧化碳。 </li><li>吸过的文化传媒和冲洗EC单层用10毫升HBSS </li><li>关闭吸HBSS和加入2ml 0.5％胰蛋白酶，0.2％EDTA溶液。在室温下孵育约5分钟。 </li><li>当乳油可以通过抽头到烧瓶的一侧脱落，加入5毫升大豆胰蛋白酶抑制剂和与吸管喷出烧瓶的表面，以进一步去除细胞。 </li><li>转移细胞到15ml试管并离心管在200×g离心5分钟。 </li><li>吸上清，悬浮颗粒欧盟在5毫升的媒体。计数细胞用血球计和每毫升乳油介质的调节细胞浓度至50,000乳油。 </li><li>分配将1ml细胞悬浮液放入24孔板含有涂覆的玻璃盖玻片上的每个孔中。孵育细胞的O / N的细胞培养孵化器在37℃下加5％的二氧化碳。 </li><li>细胞准备用于实验时汇合单层形成时，在3天内。 </li></ol> 3.准备的单核细胞<ol><li>转移的HL-60细胞，在RPMI培养在补充有10％胎牛血清，在悬浮培养，进15毫升管中。离心细胞，在200×g离心5分钟。 </li><li>除去上清液和重悬细胞在5ml介质和调节细胞浓度与媒体200万细胞/ ml。如果的HL-60荧光标记是期望的，重悬细胞在RPMI无血清和公关在第4节中描述oceed。 </li><li>添加0.5的HL-60细胞悬浮液毫升到含乳油单层的24孔簇平板的每个孔，并培育该板在细胞培养培养箱中于37℃和5％二氧化碳的时间1和3之间的选择的固定时间段小时。有点不同的是1小时和3小时的时间点，其中结合饱和度达到之间观察到。 </li><li>准备细胞清洗/收获:填写120毫升管用100毫升的HBSS。分配1毫升福尔马林成新鲜24孔板的每个孔中。 </li><li>培养结束后，使用一对锋利的钳子，拿起玻璃盖玻片从井中并保持盖玻片垂直和民建联盖玻片的边缘上的一块组织在板凳上3秒，注意不要触碰与组织盖玻片细胞覆盖表面。 </li><li>用钳子紧紧握住盖玻片，扣篮盖玻片进出的HBSS五倍。 </li><li>第五次扣篮后在HBSS中，轻拍盖玻片边缘上的组织，持续3秒。 </li><li>重复3.6和3.7中描述的扣篮和涂抹程序，合共三套5次扣篮之后轻拍。 </li><li>转移盖玻片成孔含有10％福尔马林固定细胞在RT。盖玻片准备枚举，用于长期贮存或将要进行下面描述的其他程序。 </li></ol> 4.标签HL-60细胞与细胞的跟踪（可选） <ol><li>计数和转移HL-60细胞（ 如 10元）的适量入15ml离心管中。离心机HL-60细胞，在200×g离心5分钟。去除上清液。 </li><li>重悬细胞沉淀在RPMI培养基细胞追踪无血清以1百万个细胞/ ml的浓度。孵育细胞在细胞培养孵化1小时。 </li><li>离心细胞，在200×g离心5分钟，丢弃上清液。悬浮细胞沉淀在媒体上200万℃的浓度ELLS /毫升。 </li><li>加入0.5毫升细胞跟踪标记的HL-60到生长在玻璃盖玻片每孔含有内皮细胞。继续在3.3。 </li></ol> 5.枚举胶粘剂内皮细胞<ol><li>山盖玻片DAPI反染色在显微镜载玻片上识别单个细胞。 </li><li>使用倒置显微镜用4X或10X的目标，收购的几个字段（ 例如，5场）的图像和计数的最高数字，聚集在未经照射的血管内皮细胞的单核细胞（这通常应该是3-5）。 </li><li>两次这个号码设定为单核细胞上的内皮细胞的阈值也可以是定义为一个群集。如果需要的话，不同的标准可以被用于限定根据实验的性质的集群。 </li><li>算簇的数目和内皮细胞中的场数。除以前者由后者来获得的粘合剂的内皮细胞的百分比。得分众多领域获得的计数的平均值和标准偏差。 </li></ol> 6.复染带抗体<ol><li>后在盖玻片的粘附实验及细胞已在RT固定在10％福尔马林进行15分钟，使用标准的过程10和选择的抗体若使盖玻片免疫荧光。 </li><li>添加和删​​除各种解决方案非常轻柔，避免撞出了附着于内皮细胞单核细胞。 </li></ol> 7.复染的衰老相关的β-半乳糖苷酶活<ol><li>后在盖玻片的粘附实验及细胞已在福尔马林中固定15分钟，染色为衰老相关的β-半乳糖苷酶的活性作为伴随染色试剂盒说明书所述。添加和删​​除各种解决方案非常轻柔，避免撞出了附着于内皮细胞单核细胞。 </li></ol>

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	<li>Ortega-Gomez, A., Perretti, M., Soehnlein, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+of+inflammation:+an+integrated+view.">Resolution of inflammation: an integrated view.</a> EMBO Mol Med. 5 (5), 661-674 (2013).</li><li>Libby, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+cardiovascular+disease+mechanisms.">Inflammation and cardiovascular disease mechanisms.</a> Am J Clin Nutr. 83 (2), 456S-460S (2006).</li><li>Ikuta, S., Kirby, J. A., Shenton, B. K., Givan, A. L., Lennard, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+endothelial+cells:+effect+of+TNF-alpha+on+peripheral+blood+mononuclear+cell+adhesion.">Human endothelial cells: effect of TNF-alpha on peripheral blood mononuclear cell adhesion.</a> Immunology. 73 (1), 71-76 (1991).</li><li>Watson, C., 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=IL-6+acts+on+endothelial+cells+to+preferentially+increase+their+adherence+for+lymphocytes.">IL-6 acts on endothelial cells to preferentially increase their adherence for lymphocytes.</a> Clin Exp Immunol. 105 (1), 112-119 (1996).</li><li>Sans, M., 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=VCAM-1+and+ICAM-1+mediate+leukocyte-endothelial+cell+adhesion+in+rat+experimental+colitis.">VCAM-1 and ICAM-1 mediate leukocyte-endothelial cell adhesion in rat experimental colitis.</a> Gastroenterology. 116 (4), 874-883 (1999).</li><li>Su, Y., Lei, X., Wu, L., Liu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+endothelial+cell+adhesion+molecules+P-selectin,+E-selectin+and+intercellular+adhesion+molecule-1+in+leucocyte+recruitment+induced+by+exogenous+methylglyoxal.">The role of endothelial cell adhesion molecules P-selectin, E-selectin and intercellular adhesion molecule-1 in leucocyte recruitment induced by exogenous methylglyoxal.</a> Immunology. 137 (1), 65-79 (2012).</li><li>Hallahan, D., Kuchibhotla, J., Wyble, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion+molecules+mediate+radiation-induced+leukocyte+adhesion+to+the+vascular+endothelium.">Cell adhesion molecules mediate radiation-induced leukocyte adhesion to the vascular endothelium.</a> Cancer Res. 56 (22), 5150-5155 (1996).</li><li>Vaporciyan, A. A., Jones, M. L., Ward, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+analysis+of+leukocyte-endothelial+adhesion.">Rapid analysis of leukocyte-endothelial adhesion.</a> J Immunol Methods. 159 (1-2), 93-100 (1993).</li><li>Prabhakarpandian, B., Shen, M. C., Pant, K., Kiani, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+for+modeling+cell-cell+and+particle-cell+interactions+in+the+microvasculature.">Microfluidic devices for modeling cell-cell and particle-cell interactions in the microvasculature.</a> Microvasc Res. 82, 210-220 (2011).</li><li>Lowe, D., Raj, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Premature+aging+induced+by+radiation+exhibits+pro-atherosclerotic+effects+mediated+by+epigenetic+activation+of+CD44+expression.">Premature aging induced by radiation exhibits pro-atherosclerotic effects mediated by epigenetic activation of CD44 expression.</a> Aging Cell. 13 (5), 900-910 (2014).</li></ol>

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上述的单核细胞粘附测定成功地用于设计，研究血管内皮细胞单层10的电离辐射的生物效应的实验。虽然这是不提供给评估内皮单层的密合性的唯一方法，它是使一单层即粘合剂内的比例或内皮细胞的百分比的定量的唯一方法。这是一个重要的区别是在单核细胞的粘附的全球量变，如由其他方法测量可以归因要么在内皮单层或一个亚群的内皮细胞的增加粘合性的所有小区的密合性的一般上升单层内，如图上面的例子。具有该信息的值是通过以下事实例举的能力来量化内皮细胞表现出增强的粘合性的百分比后照射导致的分级表能的结论，即这种效果不是由于特定的基因作为细胞是粘接剂的百分比的基因突变分别为大大高于（超过1000倍），该这将随机突变的基因的预期通过X射线在该剂量。 允许良好的结果的可重复性的因素是清洗制度。作为清洗方法涉及在玻璃盖玻片浸入洗涤缓冲液，接着通过涂抹在组织，避免了可变性在缓冲湍流这不可避免地产生于洗涤缓冲液到井的移液的老方法。事实上，这是过度的变异性使用迫使我们设计一个洗涤过程，从洗涤步骤中除去的变异元件移液方法获得的重复之间的观察。 诚然，这个实验谎言在需要的限制，开展单核细胞簇的人工计数，其中做Ñ加时赛允许它适于高通量分析。其次，在许多单核细胞如何构成集群的决定必须被半任意确定和电平可被设置得太高，从而导致一些真正簇的排斥。的剪切力在该方法的缺乏也可看作是一种限制在其中内皮细胞的严重增强粘附性诱导实验（ 例如，+TNFα）。在其他情况下然而，由于缺乏剪切力是有利的，因为它允许粘合性少夸张增强的检测。在单核细胞粘附性适度增加可能是特别重要的意义。 在体内 ，单核细胞不会（在典型的实验时间），预计在大批附加到内皮细胞适度增加粘附性，主要是由于剪切力。在时间然而，一些单核细胞可能会，这是更可能代表慢性炎症的环境。因此，没有剪切力是一个优势，因为它提供了实验时间框架这是典型的短，也可能转瞬即逝，让剪切应力下可以观察到适度增加粘附透露的机会很小增加内皮细胞粘附性。 该测定对其他分析，如衰老测定和免疫荧光染色后，经受了细胞的能力增加了这种方法的有效性，因为它允许特定的内皮细胞对特定细胞蛋白质或细胞状态的密合性的关联作为证明上述功能，该功能不可与旧的附着力测定。 这种方法已用于EST2永生化人冠状动脉内皮细胞和类似的结果也与原代人冠状动脉内皮细胞10获得，这表明它不是特定于只是一个特定的细胞系。此外，ADO测定血管内皮细胞的粘附性的这种方法的ption不排除使相同的细胞，以测量标记的免疫细胞的荧光标准粘附试验。总之，这份报告表明，这种方法是通用的，具有包容性，并提供比标准粘附试验更多的信息。

免疫细胞的浸润，如该行血管穿过内皮细胞层的单核细胞是在炎症1的过程中的一个重要步骤。这使得免疫细胞（免疫细胞）受伤的部位归巢。在其他实例和位置，如冠状动脉和颈动脉，通过内皮细胞层浸润的单核细胞，可导致这些细胞在动脉壁的不期望的长期停留，可能导致斑块2的形成。在免疫细胞浸润所有情况下，所述第一步骤涉及内皮细胞在血管的局部区域​​的激活。内皮细胞通过促 ​​炎细胞因子如TNF-α和IL-6，以增加细胞表面蛋白如VCAM，ICAM及E -选择其促进免疫细胞的募集和附着到内皮细胞表面的3-表达活化6。 7。该方法在精度方面的限制，导致使用放射性标记的淋巴细胞或单核细胞，接着对应于淋巴细胞或单核细胞的粘附4的放射性物质的定量。这种方法最终通过基于荧光的方法，其中感兴趣的免疫细胞进行标记荧光染料，并进行相同的程序，与上面的不同之处在于荧光代替放射性测定8取代。目前这种方法已成为最方便和由几个商业供应商试剂盒形式出售。而该测定可以测量对照和实验样本之间的粘合性的相对水平，它不会显示在荧光的变化是否是由于跨越整个电子在粘附性的均匀变化ndothelial细​​胞群，或者如果变化是由于粘合性的子细胞群内的差。这也是明显的是，洗涤的严格性上施加的结果具有深远的影响，并且更重要的是，冲洗掉不同内皮细胞单层之间未结合的单核细胞的均匀性将在结果的从重复样品之间的结果的接近程度和可重复性极大地影响。最近，几个系统即泵的单核细胞在媒体跨内皮细胞单层被用来解决这个问题9。此外，这些流动系统还概括的剪切力对内皮细胞的作用。尽管这种系统的优点是显而易见的，非常有吸引力，这也是很重要的认识到，虽然内皮细胞的粘附性，可以大大增加，如在急性炎症的情况下，受到诸如TNFα，一些其他的活化剂，如电离辐射引导学生回答10牛逼的变化帽子是不容易在体外实验的时间框架内通过这些非常严格的系统检测。虽然内皮细胞粘附性的这样的细微变化容易漏诊或驳回体外 ，它不一定是良性的体内 ，其中生活的时间，这是慢性炎症的特征内的这些小的变化，能够发挥非常显著的结果。因此，一个强大的，但敏感，和具体的方法来检测和测量内皮细胞粘合性是必要的。 在这里，我们报告直接测量内皮细胞粘附性的方法。该方法不依赖于荧光测定作为内皮细胞粘附性的间接替代指标。它揭示了变化密合性是否是由于在所有的细胞均匀变化，或只限于一个亚群。此外，它允许共染色的内皮细胞用标记物如衰老相关的β-半乳糖苷酶，细胞活力擦伤ker的，Calcien AM到抗细胞表面和胞内蛋白质，允许个别粘合剂的内皮细胞的某些细胞状态或特定的蛋白质的表达的相关性。

<table><tbody><tr><td>Hepes Buffered Saline Solution (HBSS)</td><td>Sigma</td><td>H6648</td><td></td></tr><tr><td>Glass coverslips Round 12 mm Diameter</td><td>Menzel-Glaser</td><td>CB00120RA1</td><td></td></tr><tr><td>24-well cluster plate</td><td>Costar</td><td>3526</td><td></td></tr><tr><td>Meso-Endo Cell media</td><td>Cell Applications</td><td>212-500</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Sigma</td><td>T4174</td><td></td></tr><tr><td>Soybean trypsin inhibitor</td><td>Life Technologies</td><td>17075-029</td><td></td></tr><tr><td>Cell Tracker Green</td><td>Life Technologies</td><td>C7025</td><td></td></tr><tr><td>RPMI</td><td>Sigma</td><td>R8758</td><td></td></tr><tr><td>Foetal Calf Serum</td><td>Life Technologies</td><td>10500064</td><td></td></tr><tr><td>Beta glasctosidase Assay kit&amp;nbsp;&amp;nbsp;</td><td>CellSignaling</td><td>9860</td><td></td></tr><tr><td>Fibronectin</td><td>Sigma</td><td>F0895</td><td></td></tr></tbody></table>

quantitation of endothelial cell adhesiveness in vitro

一炎症的基本过程的是免疫细胞从血管周围组织的腔的浸润。发生这种情况时内皮细胞，哪一行血管，成为粘合剂循环免疫细胞如单核细胞， 在体外测定本粘合性一直被完成通过量化单核细胞即坚持内皮层既可以作为直接计数的总数或通过粘附单核细胞的荧光的间接测量。虽然这样的测量做表明内皮细胞群体的平均密合性，它们是由许多因素，如细胞数目混淆，并没有揭示的内皮细胞，实际上是粘合剂的比例。这里，我们描述和演示的方法，它允许内皮细胞单层的测试群体内的粘合剂细胞的枚举。内皮细胞生长在玻璃盖玻片上和以下所需治疗与单核细胞（可荧光标记）的挑战。温育后，冲洗过程，涉及多轮浸没和排空后，将细胞固定。粘合剂内皮细胞，这是由单核细胞包围被容易地鉴定和列举，给予揭示的内皮细胞内的是粘合剂的人口的实际比例的粘附指数。

此方法允许在一个群体中检测个别粘合剂的内皮细胞。例如，虽然未照射的内皮细胞的单层孵化前单核细胞在周围个别内皮细胞簇受单核细胞保留几个零星的HL-60单核细胞孵育和洗涤，内皮细胞单层在后7天10Gy的辐射（ 图1）。这种现象虽然是相差显微镜下容易观察到，荧光显微镜揭示了单核细胞群的更为清晰的图像。 执行所述粘附试验后，有可能进行到使用合适的抗体用标准免疫方法染色的细胞的膜，细胞质或核（ 图2）的蛋白质。此外，基于酶的测定法，诸如衰老相关的β-半乳糖苷酶（ 图3）也可以是PErformed。 由于每个单核细胞簇对应于一个单独的内皮细胞，所述簇的枚举将揭示的粘合剂的内皮细胞的单层内的实际数目（ 图4），以及粘合剂的内皮细胞，因此在群体的百分比（ 表1，图6） 。这是唯一的方法，迄今为止，它允许内皮细胞粘附性的这样的量化。值得注意的是，在这里的实验中所用的内皮细胞是接触抑制的，实现汇合后在数目不增加。这消除了在稍后的时间点所构成的增加的内皮细胞数的潜在的复杂性。如果需要的话，连接到内皮单层的单核细胞可以通过0.5％胰蛋白酶，0.2％EDTA溶液（而不是固定在3.9）脱落并使用板读数器测定其荧光，由于是在当代方法的情况下（ 图5）。 </P&gt; <img alt="图1" src="/files/ftp_upload/52924/52924fig1.jpg" /> 图1.粘附在内皮细胞上的单层单核细胞。在未照射的内皮细胞，单核细胞粘附在零星和随机的方式单个细胞（左小图）和在集群上的后7天10格雷照射的个别血管内皮细胞单层（右图）。下部面板是顶部板的荧光图像，确认该小而明亮的球形细胞下相衬可见确实单核细胞是被预标记细胞跟踪绿色。一些单核细胞簇是由白色箭头指示。用10倍的目标图像拍摄。 <a href="https://www.jove.com/files/ftp_upload/52924/52924fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <html><img alt="图2" src="/files/ftp_upload/52924/52924fig2.jpg" /> 图2.染色血管内皮细胞的蛋白质后粘附试验的。执行所描述的粘附实验后，将细胞在玻璃盖玻片进行免疫荧光为（A） 的膜蛋白的检测（CD44），二）胞质蛋白（微管蛋白）或c）核蛋白（γ-H2AX）。的（A）和（B）（看到的20X物镜）节目单核细胞预标记与细胞跟踪绿色，右边的面板的类似图像左面板揭示CD44和微管（红色）和DAPI染色的细胞核（蓝色）。白色箭头（C）中指向该染色用抗γ-H2AX辐照内皮细胞核。这是一个染成蓝色亮用DAPI单核细胞的细胞核很容易从血管内皮细胞的椭圆形核的区别。图像用20X物镜。<html> jove.com/files/ftp_upload/52924/52924fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/52924/52924fig3.jpg" /> 内皮细胞的图3衰老相关的β-半乳糖苷酶染色。粘附试验后，将细胞在盖玻片进行染色衰老相关的β-半乳糖苷酶引起衰老细胞的溶酶体变成蓝色，因为是在内皮细胞明显由众多单核细胞，这是很容易识别的，由于其小尺寸和球形形状被选择性地结合。图片通过20X客观观察。 <a href="https://www.jove.com/files/ftp_upload/52924/52924fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 2924 / 52924fig4.jpg"/&gt; 图4.枚举单核簇上个别内皮细胞。粘附试验后，将细胞的图像取自确定了几个不同的位置和单核细胞簇和盘旋（绿色）。集群被定义为10个或更多的单核细胞对内皮细胞的集聚。单核细胞集群和12天后照射内皮细胞数量的图像中的数字（带点红色）进行计数，计算贴壁内皮细胞百分比见表1。图片通过10X物镜拍摄。 <a href="https://www.jove.com/files/ftp_upload/52924/52924fig4large.jpg" target="_blank">请点击此处查看更大的版本这个数字。</a> <img alt="图5" src="/files/ftp_upload/52924/52924fig5.jpg" /> 图5.定量粘附单核细胞中的荧光 。继阿德<html>锡永测定，将细胞在玻璃盖玻片胰蛋白酶和单核细胞的荧光测定预标记用CellTracker绿色，使用荧光板读数器，作为内皮细胞单层的密合性的间接指标。结果从这样的测量在规定的天辐照后获得，并制地图上的图形上方。 <img alt="图6" src="/files/ftp_upload/52924/52924fig6.jpg" /> 图6表示的从表粘附试验分数作为在图4中描述的和上面展示的照射是在12天后10 2Gy辐射，8％至10％的结果列1.粘合剂的内皮细胞中的三个玻璃盖玻片上五个不同的位置进行评分内皮细胞变成粘性。 <table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always"><tbody><tr><td></td>3"&gt; 胶粘剂的百分比 内皮细胞 </td></tr><tr><td></td><td>光盘1 </td><td>光盘2 </td><td>光盘3 </td></tr><tr><td> 现场1 </td><td> 8.65 </td><td> 8.29 </td><td> 8.21 </td></tr><tr><td> 现场2 </td><td> 10.44 </td><td> 7.27 </td><td> 7.21 </td></tr><tr><td> 现场3 </td><td> 9.63 </td><td> 9.05 </td><td> 8.33 </td></tr><tr><td> 4场 </td><td> 11.11 </td><td> 7.09 </td><td> 8.41 </td></tr><tr><td> 5场 </td><td> 10.55 </td><td> 9.4 </td><td> 11.61 </td></tr><tr><td> 平均 </td><td> 10.07 </td><td> 8.22 </td><td> 8.75 </td></tr></tbody></table> 表1.附着力测定分数 。如F所述胶粘剂内皮细胞的三个盖玻片五个不同的地点进行评分igure 4和结果列以上表明10 2Gy辐射后12天，照射内皮细胞的8％至10％成为粘合剂。

Watch this Scientific Journal Video about Quantitation of Endothelial Cell Adhesiveness In Vitro at JoVE.com

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

内皮细胞粘附性定量<em&gt;在体外</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

quantitation-of-endothelial-cell-adhesiveness-in-vitro

Research

JoVE Journal

Biology

Quantitation of Endothelial Cell Adhesiveness In Vitro

11.4K 次观看.  Centre for Radiation, Chemicals and Environmental Hazards, Public Health England. We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer. 

视频: Quantitation of Endothelial Cell Adhesiveness In Vitro

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

粘附频率含量为原位跨交界的分子相互作用，在细胞与细胞界面的动力学分析

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

科研

生物工程

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

到哺乳动物的上皮细胞在体外细菌粘附的含量

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

免疫与感染

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

量<em在体外</em&gt;激活的主人微血管内皮细胞在静态条件下测量的检测，以中性粒细胞粘附

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions for many of these proteins are poorly understood. Here we present a simple, rapid method for characterizing the adhesive properties of putative homophilic cell adhesion molecules. Cultured HEK293 cells are transfected with DNA plasmid encoding a secreted, epitope-tagged ectodomain of a cell surface protein. Using functionalized beads specific for the epitope tag, the soluble, secreted fusion protein is captured from the culture medium. The coated beads can then be used directly in bead aggregation assays or in fluorescent bead sorting assays to test for homophilic adhesion. If desired, mutagenesis can then be used to elucidate the specific amino acids or domains required for adhesion. This assay requires only small amounts of expressed protein, does not require the production of stable cell lines, and can be accomplished in 4 days.

Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

珠聚集测定假定的细胞粘附分子的表征

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

使用Cell-基板阻抗和活细胞成像，以衡量矩阵修改诱导实时变化的细胞粘附和去粘附

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

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 ...

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 ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

用于炎症反应的新内皮调节因子的筛选测定

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Dynamic Adhesion Assay for the Functional Analysis of Anti-adhesion Therapies in Inflammatory Bowel Disease

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a crucial step in this process and therapeutic strategies interfering with adhesion have been successfully established. The anti-&#945;4&#946;7 integrin antibody, vedolizumab, is used for the clinical treatment of Crohn&#39;s disease (CD) and ulcerative colitis (UC) and further compounds are likely to follow.
The details of the adhesion procedure and the action mechanisms of anti-integrin antibodies are still unclear in many regards due to the limited available techniques for the functional research in this field.
Here, we present a dynamic adhesion assay for the functional analysis of human cell adhesion under flow conditions and the impact of anti-integrin therapies in the context of IBD. It is based on the perfusion of primary human cells through addressin-coated ultrathin glass capillaries with real-time microscopic analysis. The assay offers a variety of opportunities for refinements and modifications and holds potentials for mechanistic discoveries and translational applications.

Dynamic adhesion of immune cells to the vessel wall is a prerequisite for gut homing. Here, we present a protocol for a functional in vitro assay for the impact analysis of anti-integrin antibodies, chemokines or other factors on the dynamic cell adhesion of human cells using addressin-coated capillaries.

动态粘附法在炎症性肠病抗粘连治疗功能分析中的作用

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a ...

内皮细胞粘附性定量

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