This study has been supported by the Collaborative Research Centre CRC TR128 "Initiating/Effector versus Regulatory Mechanisms in Multiple Sclerosis-Progress towards Tackling the Disease" (Project A9 to H.W. and C.C.G., project B1 to N.S.).

1.细胞人脑微血管内皮细胞的培养（HBMEC） <ol><li>细胞培养烧瓶的涂层<ol><li>为了制备纤连蛋白溶液，加入10毫升PBS至15mL离心管中。加入150μL纤维连接蛋白拌匀。 </li><li>以覆盖所述底部的T-25细胞培养瓶添加2毫升纤连蛋白溶液。在孵化器中孵育的细胞培养瓶为至少3个小时在37℃下。纤连蛋白包被的烧瓶可以在37℃/ 5％CO 2中可以储存2周。 </li></ol></li><li>播种和HBMEC的细胞培养<ol><li>从细胞培养烧瓶的底部吸出纤连蛋白溶液。添加7.2×10 4 HBMEC /平方厘米悬浮于6mL ECM-B培养基（= ECM-B补充有5％胎牛血清，1％青霉素/链霉素和1％的内皮细胞生长补充物）。孵育在37℃/ 5％CO 2。每天检查细胞生长用显微镜。 </li><li>改变介质每3天。收获或分裂的细胞，当HBMEC达到大约80％汇合。 HBMEC应通道1和15之间使用，以避免生理特性的损失。 </li></ol></li><li>收获HBMEC。 <ol><li>制备通过在1:1的比例混合，用PBS ACCUTASE（1×）ACCUTASE溶液:1。保持在水浴中在37℃下ACCUTASE溶液直至进一步使用。 </li><li>从细胞培养烧瓶转移ECM-B培养基至15mL离心管中。通过添加5mL PBS到细胞培养瓶的底部洗HBMEC。吸PBS和重复洗涤步骤两次以上。 </li><li>加入2毫升预热ACCUTASE溶液。孵育在37℃下2分钟。此后，HBMEC被牢牢地拍打细胞培养瓶数次重悬。细胞剥离用显微镜控制</li><li>预先存储在一个15毫升管中的ECM-B-介质只要HBMEC开始分离加回到细胞培养瓶。冲洗瓶的底部，直到大部分是HBMEC重新悬浮。 </li><li>细胞悬液转移到15毫升离心管中。离心机中以300×g于室温下10分钟。弃上清，重悬在1mL ECM-B培养基的细胞。计数细胞并稀释细胞悬浮液，以实现每毫升ECM-B培养基3×10 5的HBMEC终浓度。 </li></ol></li></ol> 2.细胞培养插入的制备<ol><li>细胞培养插入物的涂层 重要提示 :避免接触细胞培养插入的膜。 <ol><li>添加100μL的纤连蛋白溶液（参见1.1.1），以每个细胞培养插入物（ 图1A）和96孔平底平板的一个孔（光控制孔）。孵育在37℃下至少3个小时。孵育后，吸出纤维连接蛋白的解决方案。 </li><li>加入100μLHBMEC悬浮到细胞培养插入和光控制井。 600μl的ECM-B培养基添加到该单元的下部隔室培养插入。孵育3 - 4日，在37℃/ 5％CO 2，直到屏障完整性（ 图1B）到达，检查由HBMEC的显微镜评价细胞生长在光控制井。注意:不建议细胞生长超过四天。 </li><li>可选:为了模仿炎性病症抽吸从下部隔室中的培养基，并用补充有500U / mL的IFN-γ/ TNF-α前24个小时的迁移测定ECM-B培养基更换。 </li></ol></li></ol> 3.质量控制的伊文思蓝在轮回试验的当天<ol><li>伊文思蓝溶液的制备<ol><li>使用15mL离心管用200μlB27补充剂制备PBS / B27溶液混合10毫升PBS中。稀伊文思蓝原液（20毫克/毫升PBS）1:1000用PBS / B27。 </li></ol></li><li>伊文思蓝的渗透性测定<ol><li>从下部隔室，随后上部隔室吸出介质的含有汇合的单层HBMEC一个细胞培养插管。加入100μL伊文思蓝解决细胞培养插管。 </li><li>添加600μLPBS / B27到下部隔室，并在37℃/ 5％CO 2孵育60分钟。小心使用镊子取出细胞培养插管。 </li></ol></li><li>伊文思蓝测量<ol><li>从下部室中取出PBS / B27以各100μL转移到黑色聚苯乙烯96孔平底板的两个孔。在Tecan无限临M200读板器插入板和确定最佳的z位置。 </li><li>的伊文思蓝使用相应的设置测量的激发（例如:激发:620纳米，发射:680纳米，激发带宽:9 nm，发射带宽:20纳米，175x的增强，25个闪烁，积分的时间:20微秒）。 </li><li>为了确定HBMEC屏障功能比较所获取的数据与标准曲线描绘跨越HBMEC伊文思蓝渗透在种子后不同的时间点荷兰国际集团的细胞（ 图1B，右侧）。 </li></ol></li></ol> 4.迁移分析<ol><li>外周血单核细胞（PBMC）的制备。 <ol><li>加入10 mL RPMI到15mL离心管中，加入200μL的B27补充剂。计数PBMC和离心细胞以300×g离心5分钟。重新悬浮PBMC以5×10 6个细胞/ mL RPMI / B27以终浓度。 </li></ol></li><li>移民法的建立<ol><li>从下部隔室，接着含有汇合的单层HBMEC（ 图1A）的细胞培养插入物的上部隔室吸出培养基。每个供体添加每个100μL的PBMC悬浮液与细胞培养插入并且还每一个24孔板的一个孔中（ 体外控制）。 </li><li>添加600μLRPMI / B27的细胞培养插入和500微升的体外控制的PBMC的下部隔室，并在37℃/ 5％CO 2孵育6小时。 </l我&gt; </ol></li><li>迁移PBMC的收获<ol><li>使用镊子取出细胞培养插管并仔细冲洗用400微升PBS底部不接触膜。弃细胞培养插管。 </li><li>加入20μL流数荧光微（大约1,000珠/μL）向细胞培养物的下部隔室插入以及到体外控制拌匀。吸取1 mL所得PBMC悬浮液至流式细胞仪管。 </li></ol></li></ol> 5.流式细胞仪<ol><li>样品制备<ol><li>离心机PBMC在300×g下在室温下5分钟。 </li><li>通过加入荧光标记的抗体，以100μL的制备抗体溶液流式细胞仪缓冲液（PBS / 1％BSA / 2mM EDTA）中，每个样品。对于下面给出1μLCD4-FITC的结果，1μLCD3-PerCP / Cy5.5的，1μL的CD56-PC7，1μL的CD8-A700，和1μlCD16-A750每个样品使用。 </li><li>重新暂停PBMC在100μL的抗体溶液中，并在4℃下温育30分钟。 </li><li>加入250μL流式细胞仪缓冲液中并离心，在300×g离心5分钟。 </li></ol></li><li>样品采集<ol><li>以所需的量再悬浮PBMC（取决于流式细胞仪中使用的流）流式细胞仪缓冲液中。 </li><li>使用流式细胞仪用525和700nm的波长之间的有源检测器，检测流数荧光微获得染色PBMC（激发488 nm，发射525 - 700纳米）。 （下面的步骤是，如果使用GALLIOS流式细胞仪用克鲁札G软件操作的示例:（1）启动计算机（2）当操作系统被完全加载时，通过按"上流式细胞仪"按钮血细胞计数开始流动通过按下"开放协议"按钮。（3）装入相应的获取协议。（4）选择所需的协议并选择"打开"。（5）由与右点击复制为每个样本的协议在协议中虚拟转盘和在球场上"重复"左击鼠标可见按钮。 （6）标签在样品列表中的每个样本。 （7）传送所述样本以转盘的指示位置和开始采集。） </li></ol></li><li>样品分析<ol><li>使用相应的软件开所得的流式细胞术数据。确定的来自体外对照孔为transmigrated PBMC兴趣以及细胞亚群的数目，并使用相应的分析软件计数流动荧光微。 （门控策略的一个例子在结果部分中给出（ 图1 C:为了分析NK细胞亚群的轮回，首先在一个侧向散射信道（SSC）相对于前向散射信道（FSC）情节选择淋巴细胞淋巴细胞是。然后在CD3对CD56情节和CD56 + CD3显示- NK细胞被选择为NK细胞亚群之间进行区分，NK细胞都显示在一个CD56与CD16的情节和CD56 明亮的 CD16 暗/ -以及CD56 暗淡 CD16 + NK细胞被选择。此外，流数荧光微是从FSC相对于SSC情节选择并随后在信道的带525个700纳米与时间的关系，以确定它们的数目之间的发射的曲线图显示。） </li><li>来计算每个样品的总细胞数，使用流数荧光微正常化的细胞的检测数: <img alt="方程" src="/files/ftp_upload/55390/55390eq1.jpg" /></li><li>确定迁移的细胞作为总迁移的细胞和总细胞在体外控制之间比率的百分比。 </li></ol></li></ol>

The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: A.S.-M. and U.B. have no financial disclosures. T. S.-H. received travel and conference expenses from Biogen. N.S. received speaker and advisory board honoraria from Biogen and Novartis Pharma, as well as travel expenses from Biogen. H.W. received compensation for serving on Scientific Advisory Boards/Steering Committees for Bayer Healthcare, Biogen, Merck Serono, Novartis, and Sanofi-Genzyme. He also received speaker honoraria and travel support from Bayer Vital GmbH, Bayer Schering AG, Biogen, CSL Behring, Fresenius Medical Care, Glaxo Smith Kline, GW Pharmaceuticals, Lundbeck, Merck Serono, Omniamed, Novartis, and Sanofi-Genzyme. He received compensation as a consultant from Biogen, Merck Serono, Novartis, and Sanofi-Genzyme. H.W. received research support from Bayer Vital, Biogen, Genzyme, Merck Serono, Novartis, Sanofi-Aventis Germany, and Sanofi US. C.C.G. received speaker honoraria and travel expenses for attending meetings from Genzyme, Novartis Pharma GmbH, and Bayer Health Care.

在这里，我们提出了一种技术，调查淋巴细胞跨越人类血脑屏障的轮回。 在淋巴细胞迁移至CNS的体外分析是很重要的学习淋巴细胞渗出，潜在的疾病相关的变化，以及新的治疗方法的基本过程。 血 - 脑屏障模型的若干修改是可能的。例如，从上室细胞可以解析，调查了未迁移细胞群的组合物。此外，治疗的HBMEC单层与测定前24小时IFN-γ和TNF-α的可以用来模仿发炎的血-脑屏障，以研究炎性CNS疾病21,25的效果。同样，治疗HBMEC或淋巴细胞与其他物质可以调查其对淋巴细胞渗出的影响（ 例如其31上的粘附分子作用）30。某些粘附分子的参与可以使用阻断抗体对整联或它们的配体32进行研究。此外，这里介绍的实验装置允许从趋化因子或星形胶质细胞或其它细胞33,34的上清液趋化作用分析。与原代人脑源性上皮细胞HBMEC的替换扩大了这个实验设置为血-脑脊液屏障15的调查的频谱。 HBMEC不应该与其他器官衍生保持模型的CNS特异性永生化的内皮细胞或细胞所取代。然而，来自其他物种的脑衍生内皮细胞可能被用来分析轮回在各动物35。此外，视黄酸或HYDRocortisone已经描述以增加屏障功能，并可能因此被采用36,37。在有限的细胞数的情况下，进行到测定细胞的量可能会减少，因为我们的模型提供PBMC（数据未显示）2和1之间×10 5×10 6的直链回收率。要分析以下轮回罕见的细胞群，可能为了获得流式细胞仪所需的足够的细胞是必要的游泳池细胞多口井。最后，我们的实验装置也可用于药物输送研究进入中枢神经系统38。为3μm孔径通常用于允许淋巴细胞轮回，而0.4μm的孔径防止淋巴细胞轮回，但允许与研究药物输送39，40，41，42。 <p clasS ="jove_content"&gt;尽管这里所描述的实验装置的各种应用，有几点必须要牢记，以确保有意义的结果。多孔膜以及所述HBMEC单层的完整性是关键的。因此，它是强制性的，以避免膜的物理损坏，例如用移液管尖端。应在不直接接触来进行流体或细胞悬浮液至细胞培养插管的上部的加成。液滴可能在细胞培养插管的边界被擦掉。吸管尖端的水平放置确保了膜，当近腔膜在迁移测定结束漂洗的保护。的HBMEC单层的质量可以通过汇合的显微镜评价以及伊文思蓝渗透被分析为证明了血脑屏障完整性的啮齿动物实验43的评估。在低的G力和早期通道的使用HBMEC的离心分离（ 即 </eM&gt; 1 - 15）确保了单层的质量和均匀性。为了防止多孔膜溶解，坚持愿望，除了媒体所描述的顺序，与HBMEC单层工作时是非常重要的。 尽管遵守这里所描述的协议保证有意义和可再现的结果，这种技术具有一定的局限性。首先， 在体内血脑屏障是由许多细胞，其以各种方式进行交互和通过影响紧密连接1的形成加强屏障功能的形成。因此，虽然这血脑屏障模型是在体内的状况很近似，重要的环节缺失。此外，淋巴细胞外渗到跨越血 - 脑屏障的CNS是一个多步骤过程。虽然每一个步骤可以单独研究，这里介绍的技术不提供关于谁的信息剪切力2，44，45，46的影响下外渗文件过程。最后，从PBMC迁移细胞的分析可能取决于感兴趣的细胞的频率是具有挑战性的，因为细胞通常只有个位数的频率轮回。因此，感兴趣的跨多个细胞培养插入迁移的细胞的测定法或池之前的种群的分离可能是必要的。白细胞迁移到中枢神经系统的分析，其他型号涵盖一些方面在我们的模型中失踪。共培养与星形胶质细胞，周细胞和/或神经元被用来模拟血-脑屏障更好地47的复杂性。模型，包括剪切力如DIV-BBB反映多个生理条件，从而，使淋巴细胞血细胞渗出48的更复杂的分析。 </p&gt; 综上所述，我们提出适合定性和定量淋巴细胞血细胞渗出的跨越人类血脑屏障调查一个方便的技术。

从血液进入组织的细胞迁移是免疫监视的关键。特定分子相互作用的序列确保位点特异性外渗到小肠，皮肤，淋巴结，中枢神经系统（CNS），以及其他组织1。在淋巴细胞迁移的改变都参与了许多的广泛传播疾病2的病理生理学。迁移到免疫豁免CNS是被严格调控，因此，该过程的改变参与CNS相关的疾病，如脑脊髓炎3，视神经，中风和多发性硬化（MS）2，4，5，6，7。因此，研究淋巴细胞渗出，以更好地了解疾病的病理生理机制，并制定一个工具，它是非常重要的<html>疾病负担8，9，10，11，12的土壤改良。 淋巴细胞通过不同的路线迁移进入CNS。外渗通过毛细血管小静脉成可经由脉络丛内和跨血脑屏障血-脑脊液屏障蛛网膜下腔已经描述1，13，14，15。跨过血-脑屏障迁移是由淋巴细胞的内皮细胞14的相互作用进行。与此相反，以内皮细胞在外围，CNS的内皮细胞表达大量紧密连接的分子，从而严格限制细胞和蛋白质能够穿过血脑屏障的量小姑娘= "外部参照"&gt; 16。炎症导致紧密连接的松动，并且诱导的粘附分子的表达;因此，增强淋巴细胞迁移进入CNS 1，17，18。 通过血 - 脑屏障外渗是一个多步骤过程。淋巴细胞系链对内皮细胞，然后沿着主要由选择素1，15介导的过程的内皮上滚动。随后，在淋巴细胞上表达由内皮分泌的趋化因子和相应的趋化因子受体之间的相互作用诱导整联的构象变化，从而促进牢固粘附到内皮细胞1。最后，无论是淋巴细胞针对沿着血流内皮屏障抓取transmigrating进入血管周围空间中之前，或立即和直接传动和失速igrate在牢固粘附1，19，20的部位。淋巴细胞外渗所有这些步骤都可以在体外使用不同技术21进行分析。延时录像显微镜用于研究的初步圈养及压延15。粘附分析提供关于牢固停滞的详细信息内皮障碍22。如这里演示轮回测定允许免疫细胞轮回21，23，24，25，26，27，28，29的分析。 使用体外血脑屏障模型的人，我们可以最近表明，较高的MIGRCD56 明亮的 CD16的atory容量暗淡/ - NK细胞相比，他们的CD56 暗淡 CD16 +同行通过在鞘内室21这NK细胞子集的优势体现。因此，我们的实验装置似乎是适用于模拟体内的情况。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>14190-094</td>
			<td>without CaCl2 or MgCl2</td>
		</tr>
		<tr>
			<td>Fibronectin 1mg/mL</td>
			<td>Sigma</td>
			<td>F1141-5MG</td>
			<td>from bovine plasma</td>
		</tr>
		<tr>
			<td>T-25 cell culture flask</td>
			<td>Greiner BioOne</td>
			<td>690160</td>
			<td></td>
		</tr>
		<tr>
			<td>HBMEC</td>
			<td>ScienCell</td>
			<td>1000</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Pelobiotech</td>
			<td>PB-H-6023</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Sigma</td>
			<td>A6964-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>ECM-b</td>
			<td>ScienCell</td>
			<td>1001-b</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>ScienCell</td>
			<td>1001-b</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>ScienCell</td>
			<td>1001-b</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial cell growth supplement</td>
			<td>ScienCell</td>
			<td>1001-b</td>
			<td></td>
		</tr>
		<tr>
			<td>Transwell</td>
			<td>Corning</td>
			<td>3472</td>
			<td>clear, 6.5mm diameter, 3.0&#181;m pore size</td>
		</tr>
		<tr>
			<td>96-well flat bottom plate</td>
			<td>Corning</td>
			<td>3596</td>
			<td></td>
		</tr>
		<tr>
			<td>Evans blue</td>
			<td>Sigma</td>
			<td>E2129-10G</td>
			<td>stock solution: 1 g/50 mL PBS</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>50x concentrated</td>
		</tr>
		<tr>
			<td>Infinite M200Pro</td>
			<td>Tecan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well black flat bottom plate</td>
			<td>Greiner BioOne</td>
			<td>675086</td>
			<td></td>
		</tr>
		<tr>
			<td>48-well plate</td>
			<td>Corning</td>
			<td>3526</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Gibco</td>
			<td>61870-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Count Fluorospheres</td>
			<td>Beckman Coulter</td>
			<td>7547053</td>
			<td></td>
		</tr>
		<tr>
			<td>Na-EDTA</td>
			<td>Sigma</td>
			<td>E5134</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Gallios 10-color flow cytometer</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kaluza 1.5a</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TNF-&#945;</td>
			<td>Peprotech</td>
			<td>300-01A</td>
			<td></td>
		</tr>
		<tr>
			<td>IFN-&#947;</td>
			<td>Peprotech</td>
			<td>300-02</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3-PerCP/Cy5.5</td>
			<td>Biolegend</td>
			<td>300430</td>
			<td>clone UCHT1</td>
		</tr>
		<tr>
			<td>CD56-PC7</td>
			<td>Beckman Coulter</td>
			<td>A21692</td>
			<td>clone N901</td>
		</tr>
		<tr>
			<td>CD16-A750</td>
			<td>Beckman Coulter</td>
			<td>A66330</td>
			<td>clone 3G8</td>
		</tr>
		<tr>
			<td>CD4-FITC</td>
			<td>Biolegend</td>
			<td>300506</td>
			<td>clone RPA-T4</td>
		</tr>
		<tr>
			<td>CD8-A700</td>
			<td>Beckman Coulter</td>
			<td>A66332</td>
			<td>clone B9.11</td>
		</tr>
	</tbody>
</table>

analysis of lymphocyte extravasation using an in vitro model of the human blood-brain barrier

淋巴细胞外渗到中枢神经系统（CNS）是免疫监视的关键。淋巴细胞渗出的疾病相关的变化，可能会导致在中枢神经系统的病理生理改变。因此，淋巴细胞迁移的调查中枢神经系统重要的是要了解炎性中枢神经系统疾病和开发新的治疗方法。在这里，我们提出了人类血脑屏障的体外模型来研究淋巴细胞渗出。人脑微血管内皮细胞（HBMEC）的汇合生长在多孔聚对苯二甲酸乙酯的Transwell插入到模拟血脑屏障的内皮。屏障功能是由紧密连接免疫组织化学，跨内皮电阻（TEER）的测量以及伊文思蓝渗透的分析验证。这种模式使罕见的淋巴细胞亚群血细胞渗出的调查，如CD56 亮 CD16 暗淡/ - NK细胞。 Furtherm矿石，其他细胞，细胞因子和趋化因子，疾病相关的改变，以及对淋巴细胞迁移能力不同治疗方案的效果进行研究。最后，炎性刺激对内皮屏障的影响，以及不同的治疗方案可以被分析。

示出NK细胞和使用人的血脑屏障模型（ 图1A）的T细胞亚群的轮回代表性的结果示。的HBMEC单层的完整性是由紧密连接分子ZO-1，跨内皮电阻（TEER）的测量，和伊文思蓝渗透（ 图1B）的染色验证。以下3 -第4天的培养HBMEC表达的紧密连接分子ZO-1（ 图1B，左）。此外，HBMEC增长在表现出跨内皮电阻（ 图1B中）以及降低的渗透为伊文思蓝单层（ 图1B，右侧）。 HBMEC单层被用来研究NK细胞的轮回包括CD56 明亮的 CD16 暗/ -和CD56 暗 CD16 + NK细胞亚群和T细胞，包括CD4 +和CD8 + T细胞作为两个前amples（ 图1D + E，分别地）。迁移的细胞的百分比是基于作为内部对照（ 图1C）通过流式细胞术并使用归一化的流数荧光微得到的细胞计数来计算。的HBMEC单层刺激的IFN-γ和TNF-α24小时之前测定，以模仿炎性病症。细胞因子刺激导致增加的所有分析淋巴细胞群的迁移。这可能是由于包括ICAM-1粘附分子的表达增加（数据未显示）。 CD56 明亮的 CD16 暗/ - NK细胞表现出更高的迁移能力时相比，他们的CD56跨两个刺激（10.88％ 对 0.86％）和IFN-γγ/刺激的TNF-α 变暗 CD16 + NK（18.22％ 对 2.94％） HBMEC（ 图1D）。然而，轮回的炎症的结果的相对增加是为CD56 暗淡 CD16 +高</s向上&gt; NK细胞亚群（+ 342％ 对 + 167对于CD56 明亮的 CD16％ 暗淡/ - ）。这些结果模拟体内观察结果CD56 亮 暗 CD16 / - NK细胞在鞘内隔室充实。因此，血脑屏障模型似乎是合适的分析稀有淋巴细胞群的免疫细胞血细胞渗出的基本原理进入CNS 21。最后，CD4和CD8 T细胞亚群的transmigratory容量显示（ 图1E）。 <img alt="图1" src="/files/ftp_upload/55390/55390fig1.jpg" /> 图1:横跨未发炎和发炎HBMEC单层NK细胞亚群的微分迁移。 A. transwell插入（左）和实验装置（右）的图示的图象。 B.的HBMEC屏障功能验证。左:紧密连接摩尔的免疫组化染色ecule ZO-1使用兔抗人ZO-1（Abcam公司，1:200）和山羊抗 - 兔IgG-Cy3标记（1:300）上HBMEC培养3天。中心:HBMEC的第2天与培养的第4天之间跨内皮电阻（TEER）。右:标准曲线为伊文思蓝渗透为HBMEC与（"发炎"，红色）或无（"未发炎"，黑色）的刺激为500U / ml IFN-γ和TNF-α24小时。的HBMEC（黑色箭头）的接种96小时后 - 轮回测定是72执行。 CE。从16个健康个体衍生的PBMC作为在协议部分中所述进行迁移试验。在测定之前24个小时，将细胞培养插入物的一半用刺激500 IU / mL的IFN-γ和TNF-α。收获Transmigrated细胞并通过流式细胞术分析。 C.代表性的结果为PBMC从体外对照孔（顶部）和未发炎跨越HBMEC（底部）迁移后衍生的。 NK CEL<html> LS是从总淋巴细胞门控CD3 - CD56 +细胞和进一步区分为CD56 明亮的 CD16 暗/ - （"CD56 亮 ‘）和CD56 暗淡 CD16 +（’CD56 暗 "）NK细胞亚群。基于FSC / SSC特性流计数荧光微球（"珠"）的门，并在FL3确定时间曲线图的数量。典型的计算，以确定在右侧显示迁移CD56 明亮和暗淡的 CD56 NK细胞的百分比。 D.迁移NK细胞的百分比，以及CD56 明亮和暗淡的 CD56的NK细胞亚群，并迁移T细胞，包括CD4 +和以下跨越未发炎（黑色）轮回或发炎（红色）CD8 + T细胞亚群的百分比E. HBMEC描绘为平均值±SEM。 P值由学生配对t检验计算; ** P &lt;0.01，*** P &lt;0.001。/ecsource.jove.com/files/ftp_upload/55390/55390fig1large.jpg"目标=‘_空白’&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier at JoVE.com

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

使用的淋巴细胞外渗分析<em&gt;体外</em&gt;人类血脑屏障模型

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

analysis-lymphocyte-extravasation-using-an-vitro-model-human-blood

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

20.3K 次观看.  University Hospital Münster. Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro. 

视频: Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

迁移人T淋巴细胞分离，培养和分析<em在体外</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

科研

免疫与感染

Finger-stick Blood Sampling Methodology for the Determination of Exercise-induced Lymphocyte Apoptosis

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement of this phenomenon, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment of cell death. Because of this, it is difficult to determine whether reported increases in immune cell apoptosis can be contributed to the actual effect of exercise on the system, or are a reflection of the time and processing necessary to eventually obtain this measurement. In this article we demonstrate a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis. Unlike other techniques, whole blood is added to an antibody panel immediately upon obtaining a sample. Following the incubation period, red blood cells are lysed and samples are ready to be analyzed. The use of a finger-stick sampling procedure reduces the volume of blood required, and minimizes the discomfort to subjects.

Exercise is capable of inducing apoptosis in immune cells. There are various measurement limitations, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment. Demonstrated is a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis.

手指棒运动诱导淋巴细胞凋亡的测定血液抽样方法

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement ...

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

人类<em在体外承认免疫失衡的早期国家禁止作为筛检工具

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

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 ...

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 ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

使用一个调查益生素对肺炎球菌定植的影响<em&gt;体外</em&gt;粘附试验

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and Fc&#949;RI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine Fc&#949;RI&#945; was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine &#945;-chain formed a functional receptor complex with the endogenous rat &#946;- and &#947;-chains 1. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine Fc&#949;RI&#945; transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using &#946;-hexosaminidase release as a readout 2, 3. Mediator release peaked at 36.68% &#177; 4.88% at 100 ng ml-1 of antigen. This assay was modified from previous assays used to study human and canine allergic responses 4, 5. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease 6, 2, 3.

The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine Fc&#949;RI&#945; gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

的发展<em&gt;在体外</em&gt;模型研究的交互系统<em&gt;雅科仕</em&gt;<em&gt; caballus</em&gt;以其高亲和力受体了FcεRI的IgE

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

从表型分析和人体皮肤淋巴细胞分离<em&gt;离体</em&gt;细胞培养

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

一个<em&gt;体外</em&gt;血 - 脑屏障使用阻抗谱型号:对T细胞，内皮细胞的相互作用为中心

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...