我们是对TEER测量有益的讨论感谢安妮卡Engbers和弗兰克·库尔特的出色的技术支持和马库斯·舍费尔博士（nanoAnalytics有限公司）。这项工作是由德意志研究联合会（DFG），SFB1009项目A03汉王和LK，CRC TR128，项目支持A08; Z1和B01，以LK和硬件，以及跨学科临床研究中心（医学院明斯特）的授权号KL2 / 2015/14 LK。

对于所有的实验中，小鼠按照动物保健德国准则，孕育和在明斯特大学中心动物设施无特定病原体条件下维持。所有实验均根据动物实验伦理委员会的指导方针进行，北莱茵 - 威斯特法伦州，德国（LANUV，AZ 84-02.05.20.12.217）地方当局的批准。 1. MBMEC分离与培养注意:如前面详细14进行了以下修改所述隔离MBMECs: <ol><li>牺牲20只成年C57BL / 6小鼠（6-8周龄）用CO 2吸入并通过确定呼吸心跳骤停确认他们的死亡。 </li><li>冲洗，用70％的乙醇鼠标和用剪刀斩首它;删除使用镊子和剪刀头皮。使头骨的左侧和右侧切口，从枕骨大孔开始。从尾侧，德抬起头颅Ë出用镊子大脑。 </li><li>下在层流罩中，使用无菌镊子放置脑在培养皿并取出脑干，小脑和丘脑，只保留皮层。 注:显微镜使用是没有必要的任何步骤。 </li><li>放置皮质到一张无菌Western印迹纸中，用钳子轻轻摇动，直到脑膜不再可见。 </li><li>机械和酶消化（以下协议14）后，通过使用一个长的，无菌针头和5ml的柱塞收集从密度梯度内皮细胞。内皮细胞显示为红色的戒指上面红细胞一个阴暗层。这层转移20-25毫升到一个新的价值5000毫升离心管;顶部与DMEM管。 </li><li>两轮洗涤14后，悬浮在6ml含有嘌呤霉素（4微克/毫升）MBMEC介质的细胞和种子它们到一个24孔板六个涂布的孔;让他们在组织在37℃和5％CO 2培养箱中（称为从这里"孵化器"）三天。 注:有关MBMEC涂液的详细信息，请参阅材料表 。 </li><li>通过加入1ml新鲜MBMEC培养基的每孔中，而不嘌呤霉素改变介质;放板背面，在37℃和5％CO 2的两天。 </li></ol> 2.收获MBMECs <ol><li>后MBMEC隔离5天与MBMEC涂布溶液预涂可渗透刀片:添加80微升的每个刀片溶液并让它们在37℃和5％CO 2的3小时。 </li><li>仔细吸取出来的涂布溶液，并让刀片风干30分钟。加入2室温毫升（RT）的PBS中至每个孔的板，采用MBMECs洗介质;重复此步骤。添加0.05％胰蛋白酶-EDTA（每孔300微升）和留在培养箱板5-10分钟。 </li><li> 2毫升MBMEC培养基添加到每个孔中，以阻止胰蛋白酶。传送收集的细胞到15毫升离心管中并在4℃下以700 xg离心离心他们8分钟。弃上清，重悬细胞于1ml MBMEC介质的无嘌呤霉素。 </li><li>计数细胞;用0.04％台盼蓝的90微升（10倍稀释的细胞）混合10微升细胞悬浮液。移取10微升的玻璃盖和细胞计数室（血球）之间的混合。算在该腔室（N）的所有四个象限锥虫蓝 - 自由细胞和确定计数的细胞（N&#39;）的平均数目如下:N&#39;= N / 4。 </li><li>计算细胞浓度（以10 6个细胞/ ml）使用下面的公式:C = N&#39;×10 4×10，其中10 4由血球的尺寸定和10是从步骤2.4的稀释因子。 </li><li>种子2×10 4个细胞中的每个刀片260微升的体积。添加810微升MBMEC介质的每个的下部隔室以及。将与在孵化器，直到准备插入板第4条继续。 注:上下舱的体积为插入特定的，并不为所有24孔格式。 </li></ol> 3. CD4 + T细胞分离和刺激<ol><li> CD4 + T细胞分离<ol><li>通过CO 2吸入牺牲我一个成年小鼠（6-8周龄），并确定呼吸心跳骤停确认其死亡。 </li><li>放置在其上的清洁夹层板背面的小鼠并用70％的乙醇冲洗;用消毒剪刀和镊子，打开腹膜并取出脾和淋巴结（腹股沟，腋窝，肱动脉和颈部）;最后，转移的组织到含有上冰上放置5毫升的PBS 15毫升离心管中。 </li><li>下的层流罩，通过与组织滗PBS与在顶部的70微米的细胞过滤的50ml离心管中传送组织;均质所述组织通过与通过过滤器1毫升活塞加压它。 </li><li>在500 XG加入30毫升流式细胞仪缓冲液和离心机它的在4℃5分钟。弃上清，悬浮细胞在10毫升FACS缓冲。通过40微米的细胞滤网过滤悬浮液，另外20毫升流式细胞仪缓冲液添加到管中。 </li><li>离心它在500 xg离心在4℃下5分钟。弃上清，悬浮细胞在500微升FACS缓冲。 </li><li>加入20微升小鼠CD4磁珠的，拌匀孵育在4℃下15分钟。在500 XG加入25毫升流式细胞仪缓冲液和离心机它的在4℃5分钟。 </li><li>在此期间，将一个LS分离塔进磁铁并用3ml FACS缓冲冲洗。弃上清，悬浮细胞在3ml FACS缓冲。 </li><li>添加细胞到柱上。用3ml FACS洗柱缓冲液三次。从磁铁取出柱，并将其放置到一个新的15毫升CENTRIfugation管。添加5毫升流式细胞仪缓冲液中，冲洗出标记的细胞与列柱塞和离心机它在500×g离心5分钟4℃。 </li><li>弃上清，悬浮细胞在3毫升的T细胞培养基。计数细胞在2.4和种子1×10 5个细胞在100μl培养基中每孔中描述。 </li></ol></li><li> CD4 + T细胞刺激<ol><li>多克隆CD4 + T细胞刺激<ol><li>预涂层圆底96孔板用纯化的抗小鼠CD3抗体（克隆145-2C11）在PBS中，在所希望的最终浓度。例如，预涂层的完整板与α-CD3在1微克/毫升，用5毫升PBS，涡旋混合抗体的10微升（原料浓度为0.5毫克/毫升）并添加混合50微升至每个孔用多道移液器。 </li><li>留在孵化该板3小时。分离的T细胞后，用PBS洗预涂板两次。添加纯化的抗CD28抗体（克隆37.51），以在所希望的最终浓度分离的T细胞（ 例如，在1微克/毫升）;拌匀。种子的T细胞和它们留在培养箱中两到三天。 </li></ol></li><li>用树突细胞（DC）的抗原特异性CD4 + T细胞刺激 注意:如果域控制器被用作抗原呈递细胞（APC），遵循T细胞隔离协议，与这些例外情况: <ol><li>均质脾之前，用1毫升胶原酶IA型PBS中的注入它以0.5毫克/毫升，并转移到15毫升离心管中。 </li><li>孵育在水浴在37℃下15分钟。用PBS洗涤后，重悬在FACS缓冲液中的颗粒和添加20微升小鼠的CD11c磁微珠的，而不是CD4微珠。 </li><li>使用MS分离柱和合适的卷:冲洗用1ml FACS缓冲液的柱;于1ml FACS缓冲液中重悬细胞用1ml FACS缓冲液三次洗柱。 </li><li>广告选择d的抗原的DCs（ 例如 ，髓鞘少突胶质糖蛋白（MOG）氨基酸35-55在20微克/毫升），搅拌均匀，每个种子5×10 4个细胞在100μl的T细胞培养基的96圆自下而上好。 </li><li>加1×10 5个 T细胞在100μl的T细胞的培养基的每96圆底井底，如在协议对于T细胞的分离描述。 </li></ol></li><li>抗原特异性CD4 + T细胞刺激的B细胞 注:如果B细胞作为装甲运兵车，跟着T细胞隔离协议，与这些例外情况: <ol><li>从转基因小鼠，其B细胞抗原特异性（ 例如，的IgH MOG（TH）的小鼠，其B细胞特异性识别MOG 35-55）使用脾脏。 </li><li>使用抗原特异性T细胞（ 例如 ，从TCR MOG（2D2）小鼠）。加入20微升鼠标CD19磁珠，而不是CD4微珠。 </li><li>选择的抗原添加到B CELLS（ 例如 ，MOG 35-55，在20微克/毫升），搅拌均匀和种子5×10 4个细胞在100μl的B细胞培养基每96圆底井。 </li><li>在100μl的T细胞培养液中加入1×10 5个 T细胞每96圆底井，作为协议对于T细胞的分离中所描述。 </li></ol></li></ol></li></ol> 4.设置和执行测量TEER <ol><li>放置TEER仪器的24孔模块层流罩下。在使用镊子仪器MBMECs取下盖子和地方插入。 </li><li>吸管810微升新鲜培养基到模块井的下部隔室:小心添加它的插入物和模块的壁阱之间。 </li><li>关闭盖子，然后将仪器在孵化器。将仪器连接到它的计算机;打开仪器控制器上，并打开软件。 </li><li>选择在弹出的窗口中"新的测量"。车CK"显示TEER&#39;和&#39;秀CCL&#39;盒;然后按"启动"。在完成第一次测量后，选择"检查所有井"中的"结果"标签，查看所有TEER和CCL值。 </li><li>保存文件:文件&gt;另存为&gt;"文件的名称"。 注:由于TEER和CCl以自动方式被连续地测量，监视在一段三到五天它们的值。更换培养基是没有必要的，除非细胞活力是次优的，由于缺乏在TEER增加作为可视化。 </li><li>选择的时间点共同培养与T细胞MBMECs时CCL是稳定且小于1 F / cm 2，并且TEER已达到其最大水平。 </li><li>仔细检查绝对TEER和CCL的价值观和排除在MBMECs没有通过取消这种井开发的汇合单层足够的井。 </li><li>集团井的其余部分:一个良好单击鼠标右键，选择"添加以及对新孔平均值"。南Ë它，在弹出的窗口;做同样进行分组所有单个井。 </li><li>检查所有井平均以确认他们都有共同培养前相同的初始条件。如果一些有显著不同的绝对TEER值或TEER倾斜，或标准误差太大，重做分组。 注意:井的最佳分组提供TEER值的变化最小，无论是在和实验组之间。 </li><li>在"实验"选项卡，按&#39;暂停&#39;，断开仪器取出孵化器。在层流罩下取下盖子。 </li><li>制备预活化和/或幼稚T细胞，有或没有特定的细胞因子，抗体或其他物质，根据需要:用制备Ť拌匀纯化NA / LE大鼠抗小鼠IFN-γ抗体（克隆XGM1.2）:例如细胞，在每TEER仪器的井20微克/毫升。 注意:如果使用物质如颗粒酶B抑制剂，它们是一dded T细胞培养在T细胞刺激的开头: 例如 ，颗粒酶B抑制剂II（Calbiochem公司）与分离的T细胞以在DMSO 10微米的最终浓度混合。详细信息请参见材料和设备。 </li><li>删除一些从插入介质（上井室）的: 例如 ，认真吸取了150微升（删除所有媒体应该避免，因为它可能扰乱MBMEC单层）。 </li><li>添加T细胞MBMECs通过仔细吸取150微升含有2×10 5个细胞/插入物培养基。 注:在收获前激活的T细胞，只计算爆破，无台盼蓝细胞。 </li><li>关闭盖子，并把乐器回孵化器。重新连接仪器，然后按"恢复测量"。 24小时后，按"停"在"实验"标签，并保存文件（文件&gt;保存）。 </li></ol> 5.数据导出和统计分析<ol><li>通过选择文件&gt;导出的结果。 </li><li>在弹出窗口的"设置"选项卡，选择在"字段分隔符"选项，在"小数点分隔符"选项".DOT"和"制表"。 </li><li>在"导出结果"选项卡中，命名该文件，检查所有出口的油井，并勾选"TEER"和"CCL"盒子中的"选择数据"选项。 </li><li>按"导出数据，"打开导出的文件，并将数据复制到电子表格。 </li><li>正常化导出的数据（由每个重复排序，对每个运行（测量给出TEER和CCL值））:共培养之前的最后时间点的设置TEER和CCl值至100％，并相应地改变它的值对于所有其它运行时，相对于"100％"的运行。 </li><li>规范化的数据复制到所选择的软件生成的曲线图，使用各个井和显示所述平均值的标准误差为每个治疗组。 </li><li> Perform双向ANOVA统计检验用Bonferroni校正多重比较，使用所选择的统计软件。 </li></ol>

I. Kuzmanov, A. M. Herrmann, S.G. Meuth, H. Wiendl and L. Klotz have nothing to disclose. H.-J. Galla is a scientific advisor of nanoAnalytics GmbH, the manufacturer of the automated cell monitor.

所描述的协议的几个步骤是一个成功的实验是必不可少的。在初始MBMEC分离和培养，这是至关重要的工作尽可能无菌条件下进行，以防止细胞培养物的污染，真菌孢子或细菌。为了获得内皮细胞的纯培养物，推荐使用含有嘌呤霉素的前三天的介质，使内皮细胞存活，但不是其他细胞类型（特别是周细胞）41,42。值得特别注意的另一个关键步骤是添加预活化的T细胞的正确时间点的识别。期间...

血-脑屏障分开的中枢神经系统（CNS）的1系统循环- 3。它提供了一种抑制细胞的自由流动和水溶性分子的扩散，保护从病原体和潜在的有害物质的大脑的物理屏障。除了它的屏障功能，血脑屏障能使脑实质，这确保了神经元组织的适当运作的输送氧和营养物质。血脑屏障的功能特性深受其细胞和非细胞成分调节，具有高度专业化的内皮细胞是其主要结构件。血脑屏障的内皮细胞是由紧密连接（TJ）配合，缺乏穿孔的，极低的胞饮的活性，并永久主动转运机制的存在表征。嵌入内皮细胞，星形胶质细胞最终脚=相关比肩BBB的其他组成部分的EC基底膜，周细胞与神经元和小胶质6和一起形成神经血管单元（NVU），这使得能够在CNS 7适当运作- - enchymal基底膜也有助于血脑屏障2,4开发，维护和功能9。 在多种神经性疾病，如神经变性，炎症或传染病的，血脑屏障的功能受到损害2,5,10。 TJ配合物和分子运输机制的失调导致增加的血脑屏障通透性，白细胞外渗，炎症和神经元损伤。为了研究这种病理生理条件下的BBB特性， 各种体外 BBB模型已经建立9,11,12。他们共同提供了宝贵的见解屏障的完整性，透气性以及传输机制的变化。这些模型使用人，小鼠，大鼠，猪或b的内皮细胞羊产地13 - 18;原代内皮细胞或细胞系中培养或者作为单一或一起，以便在体内 19更接近地模拟血脑屏障周细胞和/或星形胶质细胞- 25。近年来，跨内皮电阻（TEER）的测量变得评估内皮屏障性能26,27被广泛接受的工具。 TEER反映阻抗跨细胞单层的离子通量及降低提供损害内皮屏障完整性并因此增加渗透性的敏感量度。各种TEER测量系统已经开发，包括上皮Voltohmmeter（EVOM），电细胞-基底阻抗传感（ECIS），以及实时细胞分析15,28 - 30。 TEER反映至（旁路线）相邻内皮之间的离子通量的电阻和成正比的屏障的完整性。在阻抗谱27,31，复合总阻抗（Z）的测定，其提供了关于通过测量CCL的屏障完整性的其他信息。 CCL涉及通过细胞膜（跨细胞途径）的电容电流:细胞层的作用就像在等效电路的电容器，分离在膜的两侧的收费及成反比的屏障完整性。当插入渗透性增加，内皮细胞附着，增殖，扩散到微孔膜。此抵抗刀片的背景电容电流（其本身的作用就像一个电容器），并直到它到达其最低水平导致降低的电容。这之后是建立TJ的络合物密封关闭相邻内皮之间的空间。这限制了通过细胞旁路径离子通量，和TEER增加，直到它到达平台期。下炎性病症，然而，endotheli人屏障被破坏:TEER降低作为TJ络合物得到破碎和CCl增加作为插入的电容成分再次上升。 我们TEER测量采用自动化小区监控系统，32:它遵循阻抗谱的原则，并扩展了其以前的应用程序。在这里，我们描述了在体外血脑屏障模型，使阻隔性能的研究，包括与免疫细胞脑内皮的相互作用;特别是活化的T细胞。 37 -这样的病理生理条件在CNS的自身免疫疾病，如多发性硬化和其的动物模型实验性自身免疫性脑脊髓炎33观察到。这里，一个重要的步骤是穿过BBB脑炎，髓鞘特异性T细胞的轮回。这之后是其在血管周围空间以及进入脑实质激活，在那里他们招募其他免疫细胞和箱diate炎症和随后脱髓鞘1,35,38。然而，这样的T细胞和内皮细胞之间的相互作用的分子机制，血脑屏障的主要成分，不能得到很好的理解。我们的协议旨在填补这一空白，并给予新的见解对内皮细胞（ 即屏障的完整性和渗透性）在他们的直接接触和复杂的相互作用与活化的T细胞的后果。 这里描述的协议利用主鼠标脑微血管内皮细胞，生长成与微孔膜渗透插入一个单层的。内皮细胞共培养的CD4 + T细胞，其可以预先激活或多克隆或在抗原特异性的方式。用预活化，但不幼稚T细胞MBMECs的共培养诱导的TEER降低和增加CCL，它提供了MBMEC功能障碍和屏障破坏的定量量度。该技术是非侵入性的:它使用内置代替筷子电极，其防止欧共体单层主要障碍;它可以被用于监测屏障功能，而无需使用细胞标记物的。它使得以自动方式连续测量并且使两个阻挡参数（TEER和CCL）随着时间的推移同时进行独立的评估。该方法也是不够敏感不同水平的T细胞活化的和内皮这样的T细胞的作用之间进行区分。 它可以在很宽范围的官能试验中使用:在炎症过程中涉及不同的细胞因子和/或趋化因子可以添加到MBMECs和T细胞的共培养;针对关于EC或T细胞侧粘附分子阻断抗体可以使用;并且可以在T细胞引发或它们的共培养内皮细胞的过程中加入的T细胞的活化标记物或它们的溶细胞性的抑制剂。该法还对T细胞轮回有用测定法，因为它可以作为前加入T细胞的MBMEC单层完整性的质量控制。这一切都使得这种方法的灵活和可靠的工具来研究BBB 体外 ，重点是激活的T细胞对EC单层完整性的影响。这是特别重要的对于理解血脑屏障破坏的机制在自体免疫疾病，如MS和其动物模型EAE，其中自反应，致脑炎的T细胞穿过BBB和引起炎症和神经元损伤的发病机理。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>cellZscope</td>
			<td>nanoAnalytics GmbH</td>
			<td>www.nanoanalytics.com</td>
			<td>including: 24-well Cell Module, Controller, PC with cellZscope software v2.2.2&#160;</td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Thermo Scientific</td>
			<td>www.thermoscientific.com</td>
			<td>SORVALL RC 6+; rotor F21S-8x50y; for MBMEC isolation</td>
		</tr>
		<tr>
			<td>flow cytometer</td>
			<td>Beckman Coulter</td>
			<td>www.beckmancoulter.com</td>
			<td>for analysis of T cell transmigration</td>
		</tr>
		<tr>
			<td>FlowJo7.6.5 software</td>
			<td>Tree Star</td>
			<td>www.flowjo.com</td>
			<td>for analysis of T cell transmigration</td>
		</tr>
		<tr>
			<td>Oak Ridge centrifuge tubes, PC</td>
			<td>Thermo Fisher Scientific</td>
			<td>3118-0050</td>
			<td>50 ml; for MBMEC isolation</td>
		</tr>
		<tr>
			<td>Transwell membrane inserts - pore size 0.4 &#181;m</td>
			<td>Corning</td>
			<td>3470</td>
			<td>for TEER measurement as the main readout</td>
		</tr>
		<tr>
			<td>Transwell membrane inserts - pore size 3 &#181;m</td>
			<td>Corning</td>
			<td>3472</td>
			<td>for TEER measurement as the quality control prior to T-cell transmigration assay</td>
		</tr>
		<tr>
			<td>24-well cell culture plate</td>
			<td>Greiner</td>
			<td>650 180</td>
			<td>flat-bottom; for MBMEC culture</td>
		</tr>
		<tr>
			<td>96-well cell culture plate</td>
			<td>Costar</td>
			<td>3526</td>
			<td>round-bottom; for immune cell culture</td>
		</tr>
		<tr>
			<td>QuadroMACS Separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-976</td>
			<td>for T cell and B cell isolation; supports MACS LS columns</td>
		</tr>
		<tr>
			<td>OctoMACS Separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-109</td>
			<td>for dendritic cell isolation; supports MACS MS columns</td>
		</tr>
		<tr>
			<td>Neubauer counting chamber</td>
			<td>Marienfeld</td>
			<td>MF-0640010</td>
			<td>for cell counting</td>
		</tr>
		<tr>
			<td>Cell strainer, 70 &#181;m</td>
			<td>Corning</td>
			<td>352350</td>
			<td>for immune cell isolation</td>
		</tr>
		<tr>
			<td>Cell strainer, 40 &#181;m</td>
			<td>Corning</td>
			<td>352340</td>
			<td>for immune cell isolation</td>
		</tr>
		<tr>
			<td>MACS MultiStand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td>for immune cell isolation</td>
		</tr>
		<tr>
			<td>MACS LS separation columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td>for T cell and B cell isolation</td>
		</tr>
		<tr>
			<td>MACS MS separation columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td>for dendritic cell isolation</td>
		</tr>
		<tr>
			<td>Mouse CD4 MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-049-201</td>
			<td>for CD4+ T cell isolation</td>
		</tr>
		<tr>
			<td>Mouse CD19 MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-052-201</td>
			<td>for B cell isolation</td>
		</tr>
		<tr>
			<td>Mouse CD11c MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-052-001</td>
			<td>for dendritic cell isolation</td>
		</tr>
		<tr>
			<td>Collagen type IV from human placenta</td>
			<td>Sigma</td>
			<td>C5533</td>
			<td>for MBMEC coating solution</td>
		</tr>
		<tr>
			<td>Fibronectin from bovine plasma</td>
			<td>Sigma</td>
			<td>F1141-5MG</td>
			<td>for MBMEC coating solution</td>
		</tr>
		<tr>
			<td>Collagenase 2 (CSL2)</td>
			<td>Worthington</td>
			<td>LS004176</td>
			<td>for MBMEC isolation</td>
		</tr>
		<tr>
			<td>Collagenase/Dispase (C/D)</td>
			<td>Roche</td>
			<td>11097113001</td>
			<td>for MBMEC isolation</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma</td>
			<td>DN25</td>
			<td>for MBMEC isolation</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Sigma</td>
			<td>F7524</td>
			<td>for MBMEC isolation</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Amresco</td>
			<td>0332-100G</td>
			<td>for MBMEC isolation</td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>Sigma</td>
			<td>P1644-1L</td>
			<td>for MBMEC isolation</td>
		</tr>
		<tr>
			<td>DMEM (+ GlutaMAX)</td>
			<td>Gibco</td>
			<td>31966-021</td>
			<td>for MBMEC isolation and MBMEC culture medium</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Sigma</td>
			<td>P4333</td>
			<td>for MBMEC isolation and MBMEC culture medium</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>Sigma</td>
			<td>D8537</td>
			<td>for MBMEC and immune cell isolation</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H3393</td>
			<td>for MBMEC culture medium</td>
		</tr>
		<tr>
			<td>Human Basic Fibroblast Growth Factor (bFGF)</td>
			<td>PeproTech</td>
			<td>100-18B</td>
			<td>for MBMEC culture medium</td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>Sigma</td>
			<td>P8833</td>
			<td>for MBMEC culture medium; only for the first three days</td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td>for harvesting MBMECs</td>
		</tr>
		<tr>
			<td>Collagenase Type IA</td>
			<td>Sigma</td>
			<td>C9891</td>
			<td>for dendritic cell isolation</td>
		</tr>
		<tr>
			<td>Trypan Blue solution, 0.4%</td>
			<td>Thermo Fisher Scientific</td>
			<td>15250061</td>
			<td>for cell counting</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E5134</td>
			<td>for immune cell isolation</td>
		</tr>
		<tr>
			<td>IMDM + 1% L-Glutamin</td>
			<td>Gibco</td>
			<td>21980-032</td>
			<td>for T cell culture medium</td>
		</tr>
		<tr>
			<td>X-VIVO 15</td>
			<td>Lonza</td>
			<td>BE04-418Q</td>
			<td>protect from light; for B cell culture medium</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Gibco</td>
			<td>31350-010</td>
			<td>for B cell culture medium</td>
		</tr>
		<tr>
			<td>L-Glutamine (100x Glutamax)</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>for B cell culture medium</td>
		</tr>
		<tr>
			<td>mouse MOG35&#8212;55 peptide</td>
			<td>Biotrend</td>
			<td>BP0328</td>
			<td>for antigen-specific T cell activation</td>
		</tr>
		<tr>
			<td>purified anti-mouse CD3 Ab</td>
			<td>BioLegend</td>
			<td>100302</td>
			<td>clone 145-2C11; for polyclonal T cell activation</td>
		</tr>
		<tr>
			<td>purified NA/LE anti-mouse CD28 Ab</td>
			<td>BD Pharmingen</td>
			<td>553294</td>
			<td>clone 37.51; for polyclonal T cell activation</td>
		</tr>
		<tr>
			<td>Recombinant Murine IFN-&#947;</td>
			<td>PeproTech</td>
			<td>315-05</td>
			<td>for T-cell transmigration assays</td>
		</tr>
		<tr>
			<td>Recombinant Murine TNF-&#945;</td>
			<td>PeproTech</td>
			<td>315-01A</td>
			<td>for T-cell transmigration assays</td>
		</tr>
		<tr>
			<td>NA/LE purified anti-mouse IFN-&#947; antibody</td>
			<td>BD Biosciences</td>
			<td>554408</td>
			<td>clone XMG1.2; recommended final concentration: 20 &#181;g/ml</td>
		</tr>
		<tr>
			<td>Granzyme B Inhibitor II</td>
			<td>Calbiochem</td>
			<td>368055</td>
			<td>recommended final concentration: 10 &#181;M</td>
		</tr>
		<tr>
			<td>PE anti-mouse CD4 antibody</td>
			<td>Biolegend</td>
			<td>116005</td>
			<td>clone RM4-4; for analysis of T cell transmigration</td>
		</tr>
	</tbody>
</table>

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.

图1提供了用于研究的T细胞和内皮细胞之间的相互作用的体外血脑屏障模型的一般概述。该实验包括三个主要步骤。第一步是从大脑皮质原MBMECs的隔离，和它们的五天培养。当它们在细胞培养板达到汇合，MBMECs胰蛋白酶化并重新接种到可渗透插入，然后将其放置在TEER仪器。该TEER和MBMECs覆铜板连续测量和监控一段三至五天。在此期间，T细胞...

Watch this Scientific Journal Video about An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction at JoVE.com

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 ...

an-vitro-model-blood-brain-barrier-using-impedance-spectroscopy-focus

Research

JoVE Journal

Immunology and Infection

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

11.1K 次观看.  University Hospital Münster. 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.

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

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T细胞受体在小鼠T细胞的逆转录病毒转导

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

科研

免疫与感染

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

在体外生物膜的形成以及8商会幻灯片

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

移植物抗宿主病的诱导和<em在体内</em&gt; T细胞监测中的应用MHC匹配的小鼠模型

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

在体外试验 ，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

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 ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

一个<em&gt;体外</em&gt;急性脑片的少突胶质细胞定向T细胞攻击模式

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

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 ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

鼠标朴素CD4<sup&gt; +</sup&gt; T细胞分离和<em&gt;在体外</em&gt;分化为T细胞亚群

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

可视化痰生物膜发展影响使用腔室盖玻片型号

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

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

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

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.