Name: a cfse-based assay to study the migration of murine skin dendritic cells into draining lymph nodes during infection with mycobacterium bovis bacille calmette-guérin
Uploaded: 2016-10-09T15:00:00.000+00:00
Duration: 7 min 14 s
Description: Watch this Scientific Journal Video about A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-

This work was supported by Karolinska Institutet, a project grant from the Swedish Research Council VR (Dnr 2014-2794) and a KID Doctoral Grant from Karolinska Institutet (All to A.G.R). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

注:C67BL / 6小鼠用于本手稿中所示的实验。这些动物都是在MTC动物设施，卡罗林斯卡医学院，瑞典斯德哥尔摩，无特定病原体条件下的房子。动物实验是根据瑞典农业局，瑞典动物保护机构和卡罗林斯卡研究所的指示和准则进行。实验是由斯德哥尔摩北方动物伦理委员会批准。 1. CFSE股票和存储的制备<ol><li>在二甲亚砜制备5-和6-羧基二乙酸酯琥珀酰亚胺酯（CFSE）的5mM储备。涡流。免除100微升等分到无菌加入0.5ml螺丝帽微管。商店等分在-80℃。 </li></ol> 2.小​​鼠<ol><li>使用近交雄性或雌性小鼠8和12周龄之间。性别和年龄匹配的动物是优选的。确保必要的动物伦理学许可证ARE在代替实验开始前。 </li></ol> 3.动物的固定与异氟醚气体麻醉<ol><li>异氟烷装入麻醉单位。装满异氟醚10毫升气密玻璃注射器。避免引入气泡。连接注射器和液体入口与供给管和向前推进器，直到在连接管中的液体是明显只是即将进入蒸发器。 注意:不要存放在注射器不使用时，异氟醚。 </li><li>麻醉动物在感应腔异氟醚，用气流保持在约400 - 400毫升/分钟，异氟烷3.5％的浓度。 注:本机将不无气流运行。 </li><li>一旦动物是睡着时，异氟烷气体打开旋塞麻醉单元上路线掩膜片。检查掩膜片是完整的，以避免气体泄漏。气流可以保持在400毫升/分钟，ALTERNAtively降低到200毫升/分钟。降低的异氟烷浓度为2.6％。 注:麻醉效果可以增加和/或通过调节流量减少。 </li></ol> 4.注射卡介苗和恢复<ol><li>直观地确认并进行测试，如脚趾捏反射试验，以确定老鼠注射执行之前，固定/睡在异氟醚掩膜片。 </li><li>在PBS中的30微升用装有29国祥½英寸针头的注射器接种在后脚垫， 牛分枝杆菌卡介苗（BCG）的1×10 6菌落形成单位。 注:分枝杆菌种群的产生在此过程3的原始描述简要地描述，并且在其他地方6实质的细节。卡介苗也可容易地从商业来源获得。作者建议卡介苗是脉冲超声或穿过注射器破坏之前，对其inoculati团块上到小鼠体内。作为微生物的刺激给予其就业在这个协议3的原始描述，但肯定的作者鼓励其他微生物刺激的测试BCG在这里重申。 </li><li>执行上述用于注射的PBS成对照小鼠中描述的相同的过程。 </li><li>监测注射后的动物，以确认恢复从麻醉中是成功的，动物能够养活自己的注入，后足。 </li></ol> 5.注射CFSE <ol><li> CFSE注射的制备: <ol><li>解冻的-80的5mM CFSE原液℃的小瓶中。稀释在PBS中的CFSE 10倍。在注射制剂填充注射器前彻底悬浮液。 </li></ol></li><li> CFSE注入和恢复: <ol><li>重复该过程中，先前接收BCG或PBS的后足在第动物麻醉3.注资2000 0.5mM的CFSE的微升。 <br/&gt;注:CFSE的注射应执行的前一天（24小时）的计划日期牺牲。 </li></ol></li></ol> 6.腘窝淋巴结的手术切除（PLN） 注:消毒手术器械和在70％的乙醇的重复去污使用鼓励贯穿此步骤。 <ol><li>安乐死小鼠。喷用70％乙醇的动物。针脚动物在仰卧位，上的夹层板。 注:作者可通过颈椎脱位安乐死建议。 </li><li>仔细使后肢大腿垂直切口。清除肌肉和脂肪用剪刀和镊子暴露，因此切除位于深处的脂肪袋的PLN，在膝盖的空洞。 </li><li>转移PLN到0.5毫升无菌PBS中，在冰上。 </li></ol> 7.从一代PLN单细胞悬液的<ol><li>通过70微米的细胞过滤PLAC均质的PLN编在含有6孔板5毫升PBS或荧光激活细胞分选（FACS）缓冲液（含PBS 2％小牛血清（FCS），5毫乙二胺四乙酸（EDTA）和1mM叠氮化钠）。通过使用3毫升注射器的后端过滤均质。 注:叠氮化钠有毒，应谨慎处理。 </li><li>与另一个5毫升PBS或FACS缓冲液洗涤过滤器，收集在一个15毫升管的材料，并在277 xg离心（1200转，R = 172 mm）作为5分钟，4℃沉淀细胞。或者，直接使用聚丙烯均质离心管均匀PLNS。 </li><li>基于该粒料尺寸，重悬PLN悬浮在PBS或FACS缓冲液， 例如 ，一个适当的体积，200 - 1000微升。使用计数室量化从每个LN悬浮液中获得的总细胞数。使用台盼蓝排除死/死亡的细胞。 </li></ol> 8.细胞染色和流式细胞仪<ol><li> TRansfer 1 - 10×10 6细胞到5ml圆底聚苯乙烯试管中。在277 xg离心（1200转，R = 172 mm）作为5分钟，4℃用FACS缓冲液，并沉淀洗涤通过离心。 </li><li>弃上清，重悬在50沉淀- 100微升抗体混合物含0.5（见用于表面染色树突状细胞[DC]标记， 材料表 ） - 1抗鼠CD16 / CD32的微克（阻止非特异性的Fc介导相互作用）在FACS缓冲液为30 - 45分钟，在冰上。避光样本。 </li><li>温育后，在277 xg离心（1,200转，R = 172，MM）洗涤用FACS缓冲液和沉淀物的细胞离心5分钟，4℃。 </li><li>重悬的细胞在50 - 100微升FACS缓冲液。收购流式细胞仪上7-10流量数据。 注:有关流量极高的评价，我们术是指读者中继这种方法的理论和实用信息下列出版物。 </li></ol>

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The authors declare that they have no competing financial interests.

国家海洋资料中心是专门的抗原呈递细胞，可以以微生物挑战在身体表面通过获取微生物抗原和经淋巴管迁移到DLN响应。那里，树突呈现这种抗原至T细胞的驱动初级T细胞应答的方式。直流偏移的从组织到DLN过程因此非常重要的是理解一个生产性T细胞应答是如何启动的。方法测量直流偏移是在随后展开上述非常有用的。 小鼠模型被用于研究感染后卡介苗，即在皮肤注射临床活疫苗皮肤DC迁移DLN。线，卡介苗在后脚垫注射模仿接种此皮肤路由。一个简单的试验，开发，因此并入该模型来研究皮肤直流子种群从足垫皮肤排水PLN卡介苗接种部位的迁移。该协议RELI上注入荧光CFSE成先前接收的BCG相同足垫，然后后来分离该排水PLN 24小时通过流式细胞仪来分析CFSE标记的细胞的存在的ES。虽然在协议没有描述，这种设置逻辑节点也可用于通过共聚焦显微镜分析制备，研究迁移过程，如迁移细胞在LN 3的定位。此外，同时使用BCG巴斯德1173P2 3和BCG SSI 1331 14已经获得了卡介苗引发的皮肤DC的迁移类似的结果。 CFSE通常用于标记细胞在体外和细胞转移后15跟踪体内这种标记的细胞。在当前的协议虽然，它被用于直接标记的皮肤细胞在原位 。 CFSE标记的分子基础是类似于FITC，这也是一种胺 - 反应性荧光染料。然而CFSE优于FITC缀合，因为它CRE阿泰非常稳定甲酰胺债券16。此外，CFSE是高度膜渗透性和被动扩散进入细胞。一旦进入，它形成内胺（荧光），该保留在标记的小区15的缀合物。 由作者开发的基于CFSE移民法使细胞响应BCG 24小时的期限内搬迁的皮肤DLN的鉴定。一个定义的时间段内因此，它衡量急性迁移和允许一个研究不同的能力，注入刺激引发的迁移。该测定距离的FITC皮肤绘画的经典技术，其测量由一个接触增敏剂的皮下，局部应用引发了累积迁移事件不同。因此，基于CFSE迁移法可用于鉴定洄游皮肤树突或其它细胞家到PLN微生物，微生物产品或其他炎性刺激的的F注射后ootpad。的确，嗜中性粒细胞和γ;δT细胞已显示出从皮肤响应移居到DLN卡介苗17,18。事实上，在测定最近使用在共感染设置以表明一个肠道局部线虫感染可以忽略卡介苗触发皮肤DC迁移到DLN 14。 直流偏移通常之间24到72小时，在FITC皮肤画自FITC-标记的评估DC是困难画19后4至5天检测。标有区议会CFSE信号的损失是不是在这里给出的分析问题，因为移民的分析仅限于24小时;这是，作者要专门研究"一夜之间"迁移事件背后的原因。然而，有兴趣在该试验中其他各方应探讨了基于CFSE跟踪早期甚至更晚的时间点。此外，由于CFSE通过注射引入它可以在任何规定的时间给出。这种灵活性允许一个uthors评估BCG感染（ 图2C）3后第5天和第6天之间的DC偏移。注入CFSE可能限制原位标记反应访问的细胞类型。例如朗格汉斯细胞（LCS）这是驻留在皮肤的最上层的DC，表皮，不良迁移响应于卡介苗在基于CFSE迁移测定（ 图3）3。然而，FITC皮肤画，位于真皮的DC期间，表皮下的层，可以容易地标记，并迁移到DLN比的LC 20,21更快。这表明，细胞可成为荧光物质标记的，而不一定需要本地化到被施加标记溶液的皮肤层。 至于BCG感染模型中，小鼠耳接种提供了真正的皮内路由更符合BCG的临床管理作为结核疫苗线。佛另一方面otpad注射，很可能在皮内和皮下途径的组合。这就是说，它需要技巧和训练来正确地和可重复性地递送卡介苗到真皮22，因此在临床实践中它可能不总是真正皮内给予，而是皮下或两者的组合。作为接种部位的脚掌确实有过这值得一提的耳朵两个非常明显的优势。首先，免疫应答浓缩至以下一个足垫注射排水PLN和这便于响应的检查。该报告的作者发现PLN成为第一个与主排水LN足垫3。其他人伊文思蓝23注射后提出的腘窝和subiliac LNS之间分流排水。尽管如此，在问候耳，有一个以上的耳DLN小鼠和这些可能不经常存在或易于定位24。后者明确地引入了牌照上诉委员会ility在寻求调查DLN反应的研究。第二，较大的体积可以被接种到足垫 - 比耳（10 50微升）。这又减少了双方在淋巴回流引进重大变化，并具有非常小的注射量，这本身就是一个问题，当谈到接种大量分枝杆菌的工作的责任。在注射体积必须小（1-10微升）耳，甚至5微升可能改变淋巴回流的和潜在迫使注入的材料的更大的量直接进入DLN以不受控制的方式。它占注射体积是重要的。这是在实验中处理的细胞中运送细菌至LN的贡献尤其重要。在BCG足垫模型中，一些BCG可能已经达到在无细胞运输的DLN。这是基于一个可以仍然从足检测BCG的小鼠的DLN，其中细胞迁移的观察垫完全由局部注射百日咳毒素3的消融。这是否是指示卡介苗可以直接访问淋巴和在一个真正的无细胞的方式到达DLN仍有待确定，因为它不能排除的是在这种情况下，其中通过注入体积的力给定接入到淋巴管分枝杆菌。 在耳或足垫免疫一个实际考虑是，动物已经得到充分固定注射到正确和可重复的方式进行的。异氟烷气体在当前协议以抑制动物中为足垫注射制备方法中描述。对异氟烷-介导麻醉的相对快速诱导和恢复时间使其成为流行的方法，但类似于其他麻醉剂，它会影响生理，其可以与实验结果25干涉。事实上，易失性和注射麻醉药废除减少淋巴流动随意肌的运动，减轻肌张力，降低lymphangion收缩26。此外，在迁移5倍减少到DC的DLN在足垫继转移已经报道在麻醉动物27。鉴于上述情况，并且由于麻醉前的处理程序都可能引起更多的压力给动物比注射本身，这是既快速施用，并且不需要回收步骤的可能性，以充分抑制动物没有麻醉剂应加以考虑。定制足垫注射剂，其中动物能在30至40秒内迅速带到一个固定的位置，并接种在足垫鼠标阻挡器将是理想的注入在后脚垫鼠标而不改变其生理学的其他方面的过程中，并会在研究中通过淋巴管解决细胞迁移是非常有用的。 总之，本报告突出了使用该定义的24小时的窗口时监控迁移的测定他们动员到DLN期间跟踪皮肤的DC了一种新的协议。这种基于CFSE迁移测定法允许通过流式细胞术，如这里详细描述的，以及通过共聚焦显微镜3检测和游走细胞的分析。它提供了一个灵活的平台用于研究各种注射刺激和其触发的DC迁移能力，并且重要的是，可以采用跟踪不仅树突而且其他人群中不同的实验设置从皮肤转移到DLN。

在车身表面局部感知的DC微生物或他们的产品，并在此过程中通过淋巴管的排水LN（DLN）1,2动员。需要对微生物抗原运输到DLN，并以入侵微生物的免疫反应随之启动该搬迁。实际上，DC的封锁或未能从被感染的组织迁移到DLN静音T细胞反应3-5。因此，旨在跟踪在单细胞水平的迁移测定法，以帮助归咎于迁徙功能到一个给定的DC子集是有用的。 有一个庞大的身躯上调动皮肤的DC到DLN的数据。后者占多少直流偏移知识从发炎或感染的网站2。因为皮肤设有一个人口众多的DC，是在实验室老鼠实验的高度可接近表面这并不奇怪。几种技术已经从而被开发，以评估从皮肤鼠DC迁移到DLN 2。 FITC皮肤绘画是目前最常用的方法。在该测定中的荧光色素荧光素-5-异硫氰酸酯（FITC）在用丙酮和接触刺激性的混合物中制备。这种鸡尾酒施加到脱毛或剃皮肤和FITC标记的细胞的积累是反过来在DLN进行分析。迁移，也可以通过注入与荧光标记的纳米粒子或微粒，从而使在皮肤已经内的荧光体颗粒吞噬细胞的跟踪皮肤研究。淋巴管也可以插管直接从淋巴中提取迁移的DC。然而，这在技术上具有挑战性的，尤其是在小鼠。用于随后的分析所得DC的数目也是一个限制因素。 尽管对皮肤DC的迁移许多前人的研究，仍然对皮肤DC动员DLN以下vacci信息的缺乏国家与卡介苗（BCG）， 牛分枝杆菌的减毒活株用于接种反对M.肺结核 。卡介苗接种于皮肤，引起触发一个T辅助细胞1型反应的有限的感染。既该动员从BCG感染皮肤的DLN，在此过程中调节其迁移直流亚种群和的因素不完全清楚。另外，在上述的光，被开发一种简单而新颖的方法，其中所述荧光染料5-和6-羧基二乙酸酯琥珀酰亚胺酯（CFSE） 原位注入跟踪DC的迁移进入DLN 3。 C57BL / 6小鼠在用BCG的接种物和牺牲前一天的足垫注射，动物被注射在相同的足垫用CFSE高浓度。二十四小时后，处死动物并取出并进行流式细胞术制备的引流腘LN（PLN）。此法可使IDE从足垫皮肤到DLN迁移过夜的细胞（ 图1）的ntification。 此外，基因打靶小鼠可以用来帮助确定调动到DLN所需的细胞因子。使用这种分析，本报告的作者们以前发现的EpCAM 低的CD11b 高迁徙的DCs皮肤BCG运输的DLN由白介素-1受体和细胞内接头分子MyD88的3调节的过程。为从上述报告由其中使用流式细胞仪检测和分析洄游皮肤树突Bollampalli 等 3发起这种基于CFSE迁移测定中的协议，在本文中描述。的优点和本测定法的缺点进行了讨论。

<table><tbody><tr><td>Isoflurane</td><td>Baxter</td><td>1001936060</td><td></td></tr><tr><td>5- and 6-carboxyfluorescein diacetate succinimidyl ester&lt;br/&gt; (CFSE)</td><td>Invitrogen</td><td>C1157</td><td></td></tr><tr><td>Dimethyl sulphoxide (DMSO)</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Sigma</td><td>F7524</td><td></td></tr><tr><td>Bacille Calmette-Gu&amp;eacute;rin (BCG)</td><td></td><td></td><td>strain Pasteur, in house</td></tr><tr><td>Trypan blue solution</td><td>Sigma</td><td>T8154</td><td></td></tr><tr><td>EDTA</td><td></td><td></td><td>In house</td></tr><tr><td>Sodium azide</td><td>Sigma</td><td>S2002</td><td></td></tr><tr><td>PBS</td><td></td><td></td><td>In house</td></tr><tr><td>Filtered water</td><td></td><td></td><td>In house</td></tr><tr><td>BD Plastipak 3 ml syringe</td><td>BD Medical Surgical Systems</td><td>309658</td><td>alternative: 5 ml syringe&amp;nbsp;</td></tr><tr><td>6 well plates</td><td>TPP</td><td>92006</td><td></td></tr><tr><td>15 ml centrifuge tubes</td><td>TPP</td><td>91015</td><td>Polypropylene</td></tr><tr><td>5 ml round bottom tubes</td><td>BD Falcon</td><td>3272948</td><td>Polystyrene</td></tr><tr><td>Univentor 400 anesthesia unit</td><td>Univentor</td><td>8323001</td><td></td></tr><tr><td>Monojet 3/10 ml syringe</td><td>Medicarrier</td><td>8881511144</td><td>29 G x 1/2 in.</td></tr><tr><td>Sterile filter unit</td><td>TPP</td><td>99500</td><td>PES membrane&amp;nbsp;</td></tr><tr><td>EPPI-Pistill homogenizer</td><td>Schuett biotech</td><td>3.200 512</td><td>Polypropylene</td></tr><tr><td>Allegra X-30R centrifuge</td><td>Beckman Coulter</td><td>B01145</td><td></td></tr><tr><td>B&amp;uuml;rker counting chamber</td><td>VWR</td><td>631-0920</td><td></td></tr><tr><td>0.5 ml screw cap microtube&amp;nbsp;</td><td>Sarstedt</td><td>72.730</td><td>Polypropylene</td></tr><tr><td>1.5 ml Eppendorf microcentrifuge tube</td><td>VWR</td><td>700-5239</td><td>Polypropylene</td></tr><tr><td>70 micron cell strainers</td><td>VWR</td><td>734-0003</td><td></td></tr><tr><td>BD LSR-II flow cytometer, operated with FACSDiva software</td><td>BD Biosciences</td><td></td><td>Discontinued. Other Flow cytometers available, minimum detection of 6 fluorochromes needed</td></tr><tr><td>FlowJo cell analysis software</td><td>Tree Star</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Antibody&lt;/strong&gt;</td><td>&lt;strong&gt;Clone&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&lt;/strong&gt;</td><td></td></tr><tr><td>MHC-II I-A/I-E&amp;nbsp;</td><td>M5/114.15.2</td><td>BD Biosciences</td><td></td></tr><tr><td>CD11b</td><td>M1/70</td><td>BD Biosciences</td><td></td></tr><tr><td>CD11c</td><td>HL3</td><td>BD Biosciences</td><td></td></tr><tr><td>CD16/CD32 (Fc-block)</td><td>2.4G2</td><td>BD Biosciences</td><td></td></tr><tr><td>CD326/EpCAM</td><td>G8.8</td><td>Biolegend</td><td></td></tr><tr><td>CD103</td><td>2E7</td><td>Biolegend</td><td></td></tr><tr><td>CD80</td><td>16-10A1</td><td>Biolegend</td><td></td></tr><tr><td>CD86</td><td>GL-1</td><td>Biolegend</td><td></td></tr></tbody></table>

a cfse-based assay to study the migration of murine skin dendritic cells into draining lymph nodes during infection with mycobacterium bovis bacille calmette-gu&#233;rin

树突细胞（DC）是通过其获取能力和梭抗原至引流淋巴结（DLN）引发的免疫应答，部分重要。区议会的DLN的动员是复杂的，仍有感染期间完全阐明。本文中所描述的是使用一个创新的，简单的测定法，它依赖于荧光染料5-和6-羧基二乙酸酯琥珀酰亚胺酯（CFSE）足垫感染的C57BL 牛分枝杆菌卡介苗（BCG）期间跟踪DC的迁移/ 6小鼠。此法可使皮肤DC亚群的积极搬迁到排水，腘LN响应BCG的表征。该协议从那里迁徙的DCs皮肤被确定通过流式细胞卡介苗模型起源。该法是和蔼可亲的DC或其他细胞的研究和鉴定的那家腘LN微生物，它们的代谢产物或其他炎症接种后刺激在足垫，因此，研究调节这些细胞的迁移因素。

基于CFSE迁移测定（ 图1）用流式细胞仪一起使用，以检测和表征出现在PLN卡介苗后感染的足垫的CFSE标记的细胞。在PLN CFSE标记的很大一部分是在MHC-II 高的CD11c + /低细胞（ 图2A），与已迁移到皮肤DLN 11皮肤的DC一贯的发现。的频率和CFSE标记，MHC-Ⅱ 高的CD11c总数+ /低细胞在PLNS从与PBS相比喷射控制（ 图2B-C）的 BCG感染的小鼠增加了，这表明BCG增强了这些外移细胞的DLN。因为在24小时的时间内该测定措施迁移，有可能建立皮肤的DC仍然进入感染（ 图2C）的DLN后5天。这扩展了目前的模式DC的迁移，远远超出了时间表初步activati在抗原特异性CD4 + T细胞中的3兹罗提的。此外，迁徙的DCs皮肤表达共刺激分子CD80和CD86（ 图2D）的水平升高。这是皮肤外植体文化和FITC皮肤画11，支持的概念，即迁徙的DCs是活化细胞以前的意见是一致的。重要的是，这两个LN驻留的DC（MHC-II +的CD11c 高细胞）和浆的DC（MHC-II 低的CD11c 低 PDCA-1 +细胞），其通过高内皮小静脉进入发炎淋巴结和不传入淋巴管12，其中负CFSE（ 图2EF），这确实是CFSE标记主要发生在脚垫。 鉴于MHC-II 高的CD11c + /低细胞代表的不是一个而是多个DC亚群13，额外的表面标记CD103，EpCAM的（CD326）和CD11b被包括在用于流式细胞仪检测（ 材料表 ），以进一步表征迁徙的DCs（ 图3）的群体的抗体染色面板。在这样做时，EpCAM的低的CD11b 高细胞亚群被确定为响应于卡介苗感染（ 图3）的主要洄游皮肤DC亚群。 此处所示的流式细胞仪数据上的流动获得流式细胞仪配备有4激光器（488，532，640和405纳米）。数据用细胞分析软件进行分析。等多色流式细胞仪比在材料表中指定的可用于成功地检测在上述研究中提到的标志物，或其他标记物的检测，对其他类型的细胞对这一问题。对于本文描述的协议中，一个细胞仪能够检测6种不同的荧光染料是NECEssary。重要的是，用于各抗体克隆被指定（ 材料数据表 ）。需要考虑，因为这可能取决于在流式细胞仪中可用的流的激光器和检测通道荧光缀合物的选择。同样地，抗体需要被滴定以确定染色的最适当稀释。 在BCG-和PBS注射的样品群体频率进行作图，并与总细胞计数一起用于计算所定义，选通的DC亚群的绝对数。在数据组的装置的差异的显着性通过学生t检验或ANOVA酌情进行分析，P &lt;0.05的截止。离群值从分析后格拉布斯的测试离群排除。 <img alt="图1" src="/files/ftp_upload/54620/54620fig1.jpg" /> 图1.概述:卡介苗感染脚垫模型和CFSE基于迁移实验。卡介苗在C57BL / 6小鼠的后肢足垫接种。在感染后不同的时间点，处死前24小时，荧光染料CFSE在先前接受BCG相同脚垫注入。排水，腘窝淋巴结是孤立的，CFSE标记的特点是流动细胞仪。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54620/54620fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54620/54620fig2.jpg" /> 图2. 洄游皮肤DC是一个主要的人口搬迁到DLN后卡介苗脚垫感染。C57BL / 6小鼠被感染的足垫卡介苗1×10 6 CFU的。处死前二十四小时，将动物用0.5mM CFSE在相同足垫注射。腘窝淋巴结收获，均质成单细胞悬浮液并进行流式细胞术。对于卡介苗后至少1天测量，CFSE被卡介苗注射后2小时。 （A和B）地块斑马显示BCG-排水PLN感染后第3天MHCII 高的CD11c + / low细胞CFSE的主要表达。来自BCG-和PBS注射的小鼠PLN CFSE标记MHCII 高的CD11c的（C）频率和总次数+ / low细胞在不同的时间点作图。 （D）的平均荧光为CD80和CD86强度（MFI）于CFSE +和CFSE NEG MHC-II 高的CD11c + /低皮肤DCS在PLN卡介苗感染后3天内确定。 （ 五 ）皮肤内的DC表达CFSE（MHC-II 高的CD11c + /低 ），LN-居民的DC（MHC-II + CD11c的高点 ）和（F）的DC浆（MHC-II 低的CD11c 低 PDCA-1 +）卡介苗后3天用流式细胞仪测定。对于每一个实验中，使用至少5只小鼠为BCG感染组和3的PBS注射的对照。条表示平均值的标准误差。每个子集内的CFSE标记的细胞总数中Bollampalli 等人给出， 公共科学图书馆病原学 2015年，11:e1005206 3。 *表示BCG感染和PBS对照组之间有统计学差异显著。一的示出了两个独立的实验。图改编自Bollampalli 等人 ，PLoS 2015年病原学，11:。e1005206 3，与出版商的许可<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54620/54620fig2large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54620/54620fig3.jpg" /> 图3.的EpCAM 低的CD11b 高的DC是主要的皮肤DC亚群作买卖ING到DLN后卡介苗脚垫感染。C57BL / 6小鼠用BCG和CFSE注射如图2（A）斑马曲线示出选通采用的策略（顶部图）和在每个CFSE +细胞的频率，以不同的定义的子集卡介苗感染（底部小图）后的时间点。每个子集内的频率可以根据感染后的时间点而改变。卡介苗后3天的人们可以期望找到所有的LN细胞中的约0.2％，MHC-II 高的CD11c + /低细胞。这些中，约6％是CD103 +细胞。对于MHC-II 高 CD103 负栅中找到的子集，可以找到约42％的CD11b 高 EpCAM的低电池，27％的CD11b 低 EpCAM的低电池和7％的信用证。 （B）CFSE +细胞内的每个定义子集在BCG感染后不同时间点的总数量显示。对于每个experime核苷酸，被用于卡介苗感染组和3的PBS控制至少5只小鼠。条表示平均值的标准误差。 *表示相对于PBS注射的对照统计学显著差异。一的示出了三个独立的实验。图重新打印从Bollampalli 等人 ，PLoS 2015年病原学 ，11:e1005206 3，与出版商的许可<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54620/54620fig3large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

基于CFSE-试验鼠皮肤树突状细胞的迁移习进引流淋巴结在与感染<em&gt;牛分枝杆菌</em&gt;卡介苗

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

a-cfse-based-assay-to-study-migration-murine-skin-dendritic-cells

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

12.1K 次观看.  Karolinska Institutet. An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Guérin.

视频: A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Guérin

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

过敏性气道炎症在肺树突状细胞的成熟和迁移分析

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

科研

免疫与感染

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

监测树突状细胞迁移<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H磁共振成像

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

成像的CD4 T细胞间质迁移发炎真皮

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

从老鼠皮肤及引流淋巴结髓细胞分离皮下继免疫减毒活<em&gt;疟原虫</em&gt;孢子

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

分离及小鼠淋巴细胞的激活

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

实验自身免疫性脑脊髓炎中枢神经系统淋巴细胞渗透的流量细胞计量分析

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating na&#239;ve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

小鼠致耐受性树突状细胞的发育及功能表征

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...

医学

Cell Migration and Cell Adhesion Assays to Investigate Leishmania-Host Cell Interaction

Leishmania is an intracellular protozoan parasite that causes a broad spectrum of clinical manifestations, ranging from self-resolving localized cutaneous lesions to a highly fatal visceral form of the disease. An estimated 12 million people worldwide are currently infected, and another 350 million face risk of infection. It is known that host cells infected by Leishmania parasites, such as macrophages or dendritic cells, can migrate to different host tissues, yet how migration contributes to parasite dissemination and homing remains poorly understood. Therefore, assessing these parasites' ability to modulate host cell response, adhesion, and migration will shed light on mechanisms involved in disease dissemination and visceralization. Cellular migration is a complex process in which cells undergo polarization and protrusion, allowing them to migrate. This process, regulated by actin and tubulin-based microtubule dynamics, involves different factors, including the modulation of cellular adhesion to the substrate. Cellular adhesion and migration processes have been investigated using several models. Here, we describe a method to characterize the migratory aspects of host cells during Leishmania infection. This detailed protocol presents the differentiation and infection of dendritic cells, the analysis of host cell motility and migration, and the formation of adhesion complexes and actin dynamics. This in vitro protocol aims to further elucidate mechanisms involved in Leishmania dissemination within vertebrate host tissues and can also be modified and applied to other cell migration studies.

Cell Migration and Cell Adhesion Assays to Investigate Leishmania-Host Cell Interaction

Here we study implications of Leishmania-host interaction by exploring Leishmania-infected dendritic cells migration. The differentiation and infection of dendritic cells, migration analysis, and the evaluation of adhesion complexes and actin dynamics are described. This method can be applied to other host cell migration studies when infected with Leishmania or other intracellular parasite species.

Leishmania is an intracellular protozoan parasite that causes a broad spectrum of clinical manifestations, ranging from self-resolving localized cutaneous ...

阐明 T 细胞动力学:实时迁移分析方法

Real-Time In Vitro Migration Assay for Primary Murine CD8+ T Cells

The adaptive immune response is reliant on a T cell&#39;s ability to migrate through blood, lymph, and tissue&#160;in response to pathogens and foreign bodies. T cell migration is a complex process that requires the coordination of many signal inputs from the environment and local immune cells, including chemokines, chemokine receptors, and adhesion molecules. Furthermore, T cell motility is influenced by dynamic surrounding environmental cues, which can alter activation state, transcriptional landscape, adhesion molecule expression, and more. In vivo, the complexity of these seemingly intertwined factors makes it difficult to distinguish individual signals that contribute to T cell migration. This protocol provides a string of methods from T cell isolation to computer-aided analysis to assess T cell migration in real-time&#160;under highly specific environmental conditions.&#160;These conditions&#160;may help&#160;elucidate mechanisms that regulate migration, improving our&#160;understanding of T cell kinetics and providing&#160;strong mechanistic evidence that is difficult&#160;to attain through animal experiments. A deeper understanding of the&#160;molecular interactions that impact cell&#160;migration is important to develop improved therapeutics.

作者聚焦:通过实时分析 T 细胞迁移了解疾病机制

This protocol providesa method of primary murine T cell isolation and time-lapse microscopy of T cell migration under specific environmental conditions with quantitative analysis.

原代小鼠 CD8+ T 细胞的实时体外迁移测定

The adaptive immune response is reliant on a T cell&#39;s ability to migrate through blood, lymph, and tissue&#160;in response to pathogens and foreign ...

基于CFSE-试验鼠皮肤树突状细胞的迁移习进引流淋巴结在与感染

摘要

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此视频中的章节

过敏性气道炎症在肺树突状细胞的成熟和迁移分析

监测树突状细胞迁移

成像的CD4 T细胞间质迁移发炎真皮

从老鼠皮肤及引流淋巴结髓细胞分离皮下继免疫减毒活

分离及小鼠淋巴细胞的激活

实验自身免疫性脑脊髓炎中枢神经系统淋巴细胞渗透的流量细胞计量分析

小鼠致耐受性树突状细胞的发育及功能表征

Cell Migration and Cell Adhesion Assays to Investigate Leishmania-Host Cell Interaction

原代小鼠 CD8+ T 细胞的实时体外迁移测定

原代小鼠 CD8+ T 细胞的实时体外迁移测定