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>

作者什么都没有透露。

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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Hepes Buffered Saline Solution (HBSS)</td>
			<td>Sigma</td>
			<td>H6648</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips Round 12mm 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&#160;&#160;</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

In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

在使用分馏爪蟾卵提取物的体外核大会

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm ...

科研

生物学

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

成人内皮细胞集落形成祖细胞的分离和大型规模扩张

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &amp;#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

定量组织样本中的γH2AX灶

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

NanoDrop微量核酸的定量

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.	
Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.

Measuring biomarkers in complex biological samples is increasingly guiding clinical decision-making. We describe a highly sensitive method to simultaneously measure cardiac myosin binding protein-C, creatine kinase MB, and cardiac troponin I in serum samples from subjects with myocardial infarction and healthy control subjects.

电化学发光免疫法测定血清心肌肌球蛋白结合蛋白-C敏感而特异的定量方法

Biomarkers are becoming increasingly more  important in clinical decision-making, as well as basic science. Diagnosing  myocardial infarction (MI) is ...

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

在体外细胞迁移和侵袭实验

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

内皮细胞小管形成实验的<em&gt;在体外</em&gt;研究血管生成

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

在斑马鱼胚胎氧化应激分析

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

蛋白表达和共定位使用复用免疫组织化学染色和多光谱成像定量

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

发展与表征<em&gt;体外</em&gt;微血管网络和内皮的定量测量的[Ca<sup&gt; 2+</sup&gt;]<sub&gt;我</sub&gt;和一氧化氮含量

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...