SS被资助由威康信托中级临床医学研究，顺时针由威康信托计划资助。

1。 Microslide准备<ol><li>预涂层的六通道microslide与鼠尾I型胶原（RTC，在PBS中稀释1:100给予220微克/毫升的工作稀释度）。这是通过注入30微升稀释的RTC溶液直接进入通道和温育2小时，在37℃，随后洗涤三次，用PBS进行的。 </li></ol> 2。在Microslides种子细胞<ol><li>在胰蛋白酶-EDTA离解培养的人肝窦内皮细胞的汇合的T75烧瓶（HSEC）（从肝组织中分离出如先前13描述的），在PBS洗，重悬在完全培养基（人内皮3×10 6个细胞/毫升基础培养基补充了2mM L-谷氨酰胺，100 U / ml的青霉素和100μg/ml的链霉素，10％热灭活的AB人血清，血管内皮细胞生长因子10纳克/毫升和肝细胞生长因子10毫微克/毫升）。注入30微升细胞悬液可怕CTLY到每个信道。 </li><li>离开细胞在37附着1小时，在湿润的培养箱中 °下用幻灯片上的齿条5％CO 2的气氛。后使细胞附着用完全培养基（ 图1）填充在每个信道的两侧的端口。 </li></ol> 3。细胞的细胞因子刺激<ol><li>离开细胞培养箱中培养24小时。使用倒置相差显微镜评估细胞的生长。 24小时前粘附实验，通过用由肿瘤坏死因子α和干扰素γ补充的完全培养基，无论是在10纳克/毫升更换生长培养基中刺激的内皮单层细胞因子。 </li></ol> 4。外周血淋巴细胞的分离<ol><li>分离自全血的外周血淋巴细胞通过最初纯化单核组分通过密度梯度离心法在适当的细胞分离介质离心25分钟，在800×g的。孵育塑料1小时的细胞，使单核细胞的粘附。 </li><li>下面粘附于塑料吸出淋巴细胞丰富上清。洗淋巴细胞和重悬它们（通常为1×10 6个细胞/流动介质毫升（内皮基础培养基含有0.1％（V / V）牛血清白蛋白（BSA））。 </li></ol> 5。或内皮细胞与白细胞抑制剂预处理<ol><li>准备功能阻断性抗体或小分子抑制剂在血管内皮media/0.1％（V / V）牛血清白蛋白的推荐稀释度。替换完全培养基用封闭抗体/抑制剂溶液中选择的信道microslide 30分钟前，流检测。 </li><li>其中，计划趋化因子受体在淋巴细胞上的预处理，重悬​​含有0.1％（V / V）牛血清白蛋白分离白细胞的RPMI溶液孵育用百日咳毒素的200纳克/毫升到方框趋化因子受体的G蛋白偶联受体活性;交替稀释具体功能阻断性抗体或趋化因子受体的小分子抑制剂的建议稀释。白细胞孵育在37℃30分钟，然后洗净，并悬浮在流体介质。 </li></ol> 6。流量分析系统的建立<ol><li> Prewarm恒温控制透明室37 °C。该室应该有硅胶管和电子供应电子电磁阀，使细胞和媒体之间切换，几乎没有死体积的插入口。该腔室安装在倒置显微镜，以允许相衬显微镜。 图2演示了流化验室和显微镜和microslide放置在显微镜载物台上。 </li><li>预填充50毫升的玻璃注射器与鲁尔锁定用10ml无菌蒸馏水，并连接至注射器端口25厘米硅胶管的长度。在注射器泵插入。根据改变的注射器泵的提取率该microslide制造商的说明，以维持0.05帕（0.5达因/厘米2， 图3）的剪切应力。 </li><li>举两个5毫升注射器，丢弃柱塞和附加桶用硅胶管电子电磁阀的两个流端口。 <ol><li>附上12厘米硅胶管的出流阀。该阀允许一个无细胞洗涤缓冲液（内皮media/0.1％（V / V）牛血清白蛋白）和淋巴细胞悬浮液之间交替。 </li><li>通过将洗涤缓冲液在两个针筒，并确保缓冲区从一个桶通过阀门和入出流量硅胶管流动冲洗电子电磁阀。 </li><li>使用阀门开关交替到另一桶，保证了缓冲器流动，并且所有气泡被从系统中删除。从桶1拆下洗涤缓冲液并更换淋巴细胞悬液图4a。 </li></ol></li><li>连接硅tubin从经由microslide适配器的注射器泵，以一个选定的microslide信道的一个端口克。然后通过一个microslide适配器从流出阀的硅胶管连接到对面的端口。确保硅管和适配器都充满了洗涤缓冲液之前，连接以防止气泡进入系统（ 图4b）。 </li><li>一旦附着在流动系统中，将microslide在显微镜载物台上，并用夹子或带子连接，以防止移动。在显微镜下用适当的相位设置设置为10X客观在一起。确保内皮细胞单层的可视化，并在重点用目镜。确保图像可以通过其安装到显微镜的图像，从而可以将其传送到显示器和记录一个相机来捕获。 </li></ol> 7。流附着力的分析检测技术和录制<ol><li>由开始withdrawa灌注内皮细胞层，2分钟洗涤缓冲液升注射泵，以消除任何碎片或绑定封闭抗体，然后切换阀，允许有5分钟推注的白细胞溶液在0.05帕恒定壁面切应力</li><li>在白细胞丸的最后2分钟，记录沿microslide的长度10个随机领域。这使得对内皮细胞单层淋巴细胞滚动/圈养的离线分析。移动到下一个之前，记录的每个字段为约10秒，并确保记录被针对流的方向，以避免记录在同一轧制细胞两次连续快速制造。 </li><li>由阀返回到起始位置跟随白细胞丸用5分钟推注的洗涤缓冲液。在此丸剂的最后2分钟，进行第二次记录阶段通过沿microslide的长度选择至少10个随机选择的字段。移动到下一个记录之前，每个字段为约5秒，并确保内皮细胞单层贴壁leucocytes是明确的重点。这使得附着的图案离线分析。 </li></ol>

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作者宣称没有竞争经济利益

为成功地执行流分析中最关键的步骤是确保内皮细胞的健康和汇合单层准备之前的流动粘附实验。初级内皮细胞可能很难培养，并在培养方法的改变很敏感。但重要的是1）流动室被充分且均匀地涂覆有内皮细胞的单层;为HSEC我们采用鼠尾I型胶原，但是这可能不同于其他内皮种群，2）培养基是适当的细胞类型，为HSEC我们在协议部分描述了我们完全培养基。其他重要的步骤包括设置注射泵以适当的比率来反映生理水平的剪切应力。 在流量测定，有必要防止流电路，它可以破坏内皮细胞单层或由内皮表面剥离的免疫细胞内的气泡。这可以通过连接被阻止suring所有的硅胶管和适配器灌注前连接洗涤缓冲液，所有的气泡被去除，并且在使用前媒体预热。当连接到适配器上的microslide这是很重要的，有连接过程中的液/液界面的端口，如果存在任何空气，那么这将形成系统，该系统将注射器抽出时破坏内皮细胞单层中的空气间隙一步。在注射器针筒的白细胞溶液需要定期搅动以确保该细胞不沉淀，从而保持恒定的细胞密度在整个实验。 在记录步骤，它保证了内皮细胞层的图像被适当地聚焦和清晰，以允许精确的离线分析是很重要的，并且在该流量测定法（后白细胞药团）的第二步骤中有足够的时间在洗涤缓冲液中左录制前阶段重新开始，以确保所有非贴壁白细胞被删除。同样地，必须使用内皮细胞中的适当密度的单层，以防止细胞可​​以在窄的毛细管流动模式干涉的损失，也可以是硬从相差显微镜下放大粘附白细胞区分。我们已经描述了最佳接种密度对人肝窦内皮细胞，但可能不同内皮种群和物种之间变化。 显著的进步已经取得了在研究招募白细胞在动物模型与活体显微镜。流粘附试验方法的主要优点是，白细胞招募可以在一个二进制系统与原代人内皮细胞进行研究。此外，这些相互作​​用可以根据剪应力的生理相关水平进行研究。它证实活体研究发现在动物与人类蜂窝系统是重要的，因为有可能是differencES在物种之间内皮属性。一项所述的流量测定的局限性在于，白细胞招募正在研究中的内皮单层的单细胞环境。此外，一旦白细胞已粘附和transmigrated跨内皮它们可能不存在足够数量的被分离并进行下游处理。 尽管有这些限制，一旦流动粘附实验已经掌握了它可以被开发来进行白细胞黏附级联的进一步分析和适应复述一个多细胞环境。单个字段以及使用跟踪软件的延长记录可以用来分析白细胞爬行行为。此外，该流分析完成后microslides可以使用激光扫描共聚焦显微术和免疫荧光标记，研究粘附和轮回中更详细地进行讯问。此外，我们先前开发编着的体外模型，其中流过的白细胞可与肝血管内皮细胞通过肝细胞的存在条件进行交互。此法也可开发研究白细胞亚群：我们集团已与子集，如调节性T细胞，B细胞和肝脏浸润的白细胞进行研究。 这些研究的证据表明，流动粘附实验是一个功能强大的工具来研究一般和器官特异性白细胞招聘人类系统。

现在是公认的一般白细胞招聘如下多级级联的附着力1的范例。这涉及白细胞从血管内皮细胞衬里的容器壁流动的血液的采集。最初，白细胞经受这是由选择素受体或免疫球蛋白超家族的成员介导的一个轧制工序。这使得G-蛋白偶联受体（GPCRs）表达于白细胞表面由呈现在内皮糖萼的趋化因子被激活。这导致整确认的改动在白细胞表面的“高亲和力&#39;的状态，并逮捕和牢固粘附于血管内皮细胞。随后是在容器上的白细胞的形态变化和爬行牢固粘连。最后的步骤是轮回穿过内皮细胞单层，其可以通过细​​胞旁或跨细胞途径发生。 而多级级联的附着力描述s白细胞招聘的身体内的一般机制有器官特异性差异。在肝脏中的大多数白细胞招募发生在相对 ​​于其他器官，其中招募一般发生在毛细血管后微静脉2内肝窦内。肝窦是低剪切环境和白细胞进行坚定的附着力是选择素独立前2简要圈养的一步。这些通道是由肝窦内皮细胞是不连续的，并且包含窗孔，在直径开孔100-200nm的内衬，以及缺乏基底膜3。阐明介导白细胞招聘人力对面肝窦内皮细胞的分子机制，可以识别器官的炎症性肝脏疾病的具体治疗目标。 流动附着力试验正在研究白细胞招聘必不可少的工具。他们允许白细胞recru重建itment在剪应力分析下明确定义的粘附力的存在。最频繁使用的测定是研究白细胞粘附到培养的内皮单层的或纯化的底物。市售的流动室被用于灌注细胞两个平面之间的层流的条件下，然后显现粘合性的动态过程，在显微镜4。前面的组显示出，某些粘合剂的相互作用仅采取下流动的地方，无法在静态测定5,6研究。 我们已经用这种技术来概括肝窦和研究低剪切应力条件下白细胞招聘。初级人类HSEC在microslides和白细胞培养就可以在流量计算，肝窦内重现剪切应力的速度进行灌注在这个单层。的剪切应力是应用于平行或相切的面作为奥普一个应力osed正常应力相垂直。任何沿边界移动的流体将施加在该边界上的剪应力。剪切应力已被证明是淋巴细胞轮回7的基本组成部分。在这些条件下的粘附级联的每个步骤可通过相衬显微镜可观察到。这种方法已允许了重要的见解进入肝脏，包括研究常规粘附分子8，趋化因子和趋化因子受体9-11的作用，和非典型的粘附分子，如血管粘附蛋白-1（VAP-1内白细胞的募集）8,12和共同淋巴管和血管内皮受体-1（叻-1）13。虽然这个实验已经提到在我们几个组的出版物，它描述颇短，我们已经采取了这一机会，提供一步地指导了详细的步骤，以帮助故障诊断和预防技术错误时尝试ing的检测。此外，我们最近改变了microslide室的采购，它允许精确的改建剪切应力。我们相信，这拓宽了检测到其他内皮细胞和免疫细胞的适用性。下面的方法描述了制备和技术，用于执行与人类肝窦内皮细胞和外周血淋巴细胞的流程基于粘附实验。

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			<td height="21" style="height:21px;width:227px;">Name of the reagent/ Equipment</td>
			<td style="width:168px;">Company</td>
			<td style="width:195px;">Catalog number</td>
			<td style="width:447px;">Comments</td>
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			<td height="21" style="height:21px;">Six channel &#956;-slide VI 0.4 flow chamber&#160;</td>
			<td>Ibidi</td>
			<td>80601</td>
			<td>Other channel size and pre-coated slidess are available depending on assay requirements.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flow adaptors&#160; &#956;-slide VI 0.4</td>
			<td>Ibidi</td>
			<td>80646</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flow assay chamber</td>
			<td>Solent Scientific</td>
			<td>33-3322</td>
			<td>These chambers are custom made by the company dpending on the model of microscope and accessories.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inverted Microscope IX2</td>
			<td>Olympus, UK</td>
			<td>Model IX50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Harvard Syringe Pump</td>
			<td>Harvard Apparatus, UK</td>
			<td>702101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electronic solenoid valve</td>
			<td>Lee Products Limited,UK</td>
			<td>Part Number LFYA1226032H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicon Tubing large</td>
			<td>Fisher Scientific</td>
			<td>FB50855</td>
			<td>2mm Inner diameter, 4mm Outer diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicone Tubing-small</td>
			<td>Fisher Scientific</td>
			<td>FB50853</td>
			<td>1mm Inner diameter, 3mm Outer diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Harvard Glass Syringe</td>
			<td>Harvard Apparatus, UK</td>
			<td>55-0962</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell separation medium/Lympholyte</td>
			<td>VH Bio</td>
			<td>CL-5020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat Tail Collagen</td>
			<td>Sigma Aldrich</td>
			<td>C3867-1VL</td>
			<td></td>
		</tr>
	</tbody>
</table>

a flow adhesion assay to study leucocyte recruitment to human hepatic sinusoidal endothelium under conditions of shear stress

白细胞浸润到人肝组织中所有成年肝脏炎性疾病的一个共同的过程。慢性浸润可以驱动纤维化进展的发展为肝硬化。了解介导白细胞募集到肝脏的分子机制可以识别肝脏疾病的重要治疗靶标。白细胞招募期间的关键相互作用是炎症细胞与剪切应力的条件下，内皮细胞。招聘到肝脏发生这是由肝窦内皮细胞（HSEC）内衬肝窦的低剪切通道内。肝窦内的条件可以通过在特定的流速内衬由人类HSEC单层通道灌注白细胞进行概括。在这些条件下的白细胞进行一个简短的圈养步骤和随后的激活和紧贴性，其次是跨内皮爬行一步，后续的轮回层。用相衬显微镜，这&#39;粘附级联“的每一步骤，可以可视化并记录随后的离线分析。内皮细胞或白细胞可以预先用抑制剂，以确定特定的分子在此过程中的作用。

此法有可视化的多步流程的附着力和级联通过比较对照实验结果对那些与分子抑制剂阐明潜在的分子机制的能力。各种血管床可通过将特定的内皮细胞和改变剪切应力条件来概括。 粘附级联的每个步骤可以在离线状态下通过在协议中列出的记录方法进行分析。粘附级联的第一步骤是可被表示为总贴壁细胞的百分比的白细胞的滚动。离线分析允许白细胞丸剂过程中列举的每个记录的字段的贴壁细胞的数量。图像回放允许细胞，贴壁牢固和那些跨内皮经历了滚动运动的比较。滚动运动可以使用该技术进行可视化，每个字段中记录为至少10秒。滚动细胞通过在内皮细胞表面的减少速度识别相比，流动的细胞。这种行为必须证明至少5秒，而没有脱离。肝窦内的粘附级联发生在低剪切环境和体内研究已证实最少的轧制仅简要圈养步骤。我们已经证实，该流量测定反映肝血窦通过证明粘附白细胞少于10％的持续滚动激HSEC在这些测定中的环境。 粘附级联的下一步骤是牢固的粘合。总坚持可从记录的第二阶段的洗涤缓冲液丸（步骤7.3）中进行计算。离线分析允许牢固地粘附细胞的总数量，以在每个字段进行计数（ 图5）。牢固粘附的细胞被定义为细胞，静止或shapechanged缓慢爬行行为。每场的细胞的平均数量可以被计算出来。该图可以被用来在与所述视场的总表面积结合（使用确定的标线或等效物），淋巴细胞（通常为1×10 6个细胞/ ml）的浓度和流速来表达的程度淋巴细胞粘附以贴壁细胞/毫米2/10 6细胞的灌流。 学习粘连的模式涉及的最后两个步骤的附着力级联包括形状变化，爬行和跨内皮迁移的分析。白细胞粘附于HSEC单层的上表面出现相亮，而那些通过单层迁移阶段出现暗（ 图6）。该细胞可以被分类为显示出“静态”粘着（nonmigrated /轮），“形状改变”的形态或为&#39;迁移&#39;和单个类别，然后表示为的百分比胶粘剂总人口。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51330/51330fig1highres.jpg" src="/files/ftp_upload/51330/51330fig1.jpg" /> 图1。单层原代人肝窦内皮流室中的细胞。 A）充满了含有内皮细胞的单层介质开始流动粘附实验的融合内皮细胞单层B）相衬图像之前Microslides，内皮细胞应在microslide已预涂播种（人类肝脏内皮细胞，这应该是与鼠尾胶原蛋白I型），它是至关重要的内皮细胞是健康的文化和融合。 <a href="https://www.jove.com/files/ftp_upload/51330/51330fig1highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" fo:content-width="5in" fo:src=“/ files/ftp_upload/51330/51330fig2highres.jpg”SRC =“/ files/ftp_upload/51330/51330fig2.jpg”/&gt; 图2。流量测定室。一种流量测定室的建立在这里可以看到，它包括一个安装在倒置显微镜的透明腔。加热器被放置在所述腔室，并应恒温控​​制，以保持37℃的温度。应该有可从该腔室中的microslide连接硅胶管到注射器泵，其位于外侧的端口。该microslide直接放置在显微镜舞台上。 <a href="https://www.jove.com/files/ftp_upload/51330/51330fig1highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/51330/51330fig3highres.jpg" src="/files/ftp_upload/51330/51330fig3.jpg" /> 图3。注射泵 。注射器泵连接通过硅胶管的流室编。该泵被设置为根据所需的检测所需的剪切应力特定的退出率。 <a href="https://www.jove.com/files/ftp_upload/51330/51330fig1highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图4" fo:content-width="5in" fo:src="/files/ftp_upload/51330/51330fig4highres.jpg" src="/files/ftp_upload/51330/51330fig4.jpg" /> 图4。连接阀流室A）电子电磁阀允许，几乎没有死角。 乙含有两种细胞或媒体2针筒之间切换）一旦阀门被刷新和两桶的设立，从阀门的硅胶管连接到所述流动室。该连接上的硅橡胶管上的流动腔室的端口适配器时，有一个液/液界面是至关重要的。 &lt;A HREF =“https://www.jove.com/files/ftp_upload/51330/51330fig1highres.jpg”目标=“_blank”&gt;点击这里查看大图。 <img alt="图5" fo:content-width="5in" fo:src="/files/ftp_upload/51330/51330fig5highres.jpg" src="/files/ftp_upload/51330/51330fig5.jpg" /> 图5。测定白细胞总数坚持。在最后两分钟的洗涤缓冲液丸步骤（在协议中所述）的，最少十场随机应记录。这些可以脱线进行分析，并牢固地粘附细胞的总数量可以在每个字段进行计数。白细胞黏附总能控制腔和那些与预处理封闭抗体之间进行比较，在这里我们将展示一个领域的代表从控制幻灯片和预处理细胞间黏附分子-1（ICAM-1）阻断抗体的幻灯片。箭头已经添加到突出粘附白细胞，在代表性从控制幻灯片略去场共有25白细胞认定是和ICAM-1的块滑动共有13白细胞识别是。比例尺= 100微米。 <a href="https://www.jove.com/files/ftp_upload/51330/51330fig1highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图6" fo:content-width="5in" fo:src="/files/ftp_upload/51330/51330fig6highres.jpg" src="/files/ftp_upload/51330/51330fig6.jpg" /> 图6。也可以使用记录的字段的白细胞粘附于内皮细胞单层用相差显微镜模式分析。离线分析，以研究白细胞粘附的方向和速度。粘附级联的特定步骤可以可视化，并使用相位对比成像定量。相亮细胞是粘附牢固，但不激活可以被称为&#39;轮&#39;的附着力，这是激活细胞D和明亮的阶段可以称为“形状变化”的，哪些是黑暗期的细胞是已经过跨内皮迁移，可以称为“迁移”的细胞。该图像显示粘连每个模式的例子。 <a href="https://www.jove.com/files/ftp_upload/51330/51330fig1highres.jpg" target="_blank">点击这里查看大图</a> 。

Watch this Scientific Journal Video about A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress at JoVE.com

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

白细胞募集到肝脏发生这是由独特的肝窦内皮细胞衬里肝窦的专业渠道内。整个生理切应力条件下的人力肝窦内皮细胞白细胞招聘相衬显微镜可以促进其背后这一过程的分子机制的阐明。

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 ...

a-flow-adhesion-assay-to-study-leucocyte-recruitment-to-human-hepatic

Research

JoVE Journal

Immunology and Infection

15.2K 次观看.  University of Birmingham. 白细胞募集到肝脏发生这是由独特的肝窦内皮细胞衬里肝窦的专业渠道内。整个生理切应力条件下的人力肝窦内皮细胞白细胞招聘相衬显微镜可以促进其背后这一过程的分子机制的阐明。 

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

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

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

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

成像白细胞粘附到血管内皮细胞在高腔内压力

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

科研

免疫与感染

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 ...

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

分离人脐静脉内皮细胞和水流条件下其使用中的中性粒细胞轮回研究

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

一个<em在体外</em&gt;模型研究人类巨噬细胞分化和极化的异质性

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

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 ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

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 ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

静电粘附实验对整合素激活的T细胞研究

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

一种简单快速议定书非酶离解新鲜人组织的浸润淋巴细胞的分析

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

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 ...