We thank Dr. Peter T. Sage for his assistance in generating some of the representative images. This work was supported by an NIH R01 grant to C.V.C. (HL104006).

<ol>
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The authors have nothing to disclose.

总体而言，这一协议描述方法研究内皮细胞作为一）充分研究生理装甲运兵车和ii）作为"平面细胞APC模式"一种新的类型。相对于前者，它已成为日益认识到，非造血外围设备（或"基质"）的APCs发挥关键的，非冗余的作用（即，相比于造血的APC）在成形适应性免疫应答16-18。在这样的"半专业"装甲运兵车，血管及淋巴管内皮细胞（这远多于专业装甲运兵车）均名列前茅赞赏16-18。然而，信号事件的细节和它们的连接的免疫突触基本上仍未经调查。本文所描述的方法提供了在这个新兴领域前进理解的基础。例如，应用这些方法的IS形成在从已知会表现出不同的自适应的不同组织的内皮的研究Ë免疫功能（ 例如 ，肝脏和淋巴管内皮细胞）可能会特别有启发。 显著，这种方法有助于弥合专职APC和人工APC的基底模型之间的间隙，以提高询问适应性免疫应答的基本机制的能力。而平面APC基板（ 即 ，脂双层，抗体包被玻璃）提供最佳成像（这导致了许多重要见解7,11-14），这种模式本质上是有限的。可替代地，生理的细胞-细胞扫描动力学细节迄今为止已经深刻地取向有关的成像问题8-10遮蔽。在目前的方案中提供的成像能力克服这一障碍，唯一露出否则无法察觉的立体亚细胞结构和生理APC-T细胞界面的快速重构。这些报价的潜力抗原signalin的新认识G。 例如，升值T细胞形成ILPS富含的TCR，肌动蛋白和信号分子（例如， 图2D，图3），表明这些可能代表了生理三维计数器份到平面TCR信令微簇上观察到人工APC基板6,7,12-14,29-31。这意味着，在生理设置，淋巴细胞可以采用ILPS离散的亚细胞"3D反应卷"与扩大"signalosome&#39;般的性能和维持通过集中的重要分子/活动32,33信号。此外，对生物力学显著投入 （即，细胞-细胞力应用程序）在景点的存在下，如果针对ILP在APC突起可从成像推断。这是一个平凡的机械力日益被看作抗原信号的关键调解人，而恰恰力量是如何如林语堂仍不清楚14,34-37。 该模型的合理替代品专职APC的总体相关性支持我们的直接证明，类似的ILP阵列可以在形成于内皮细胞和DC（和在较小程度上的B细胞）时类似的成像方法被采用突触可见（例如，使用同样的荧光制定者和为平面取向面19）的控制权。然而，它需要考虑，EC的与专职APC膜面的相对生物力学特性是未知的，并且在该参数的差异可能会影响到IS拓扑和抗原反应的细节。 密钥在此描述的方法是强烈相关的其第好处。这又是荧光信号的强度高度相关。因此，要特别注意优化转fluores和染色是关键分记者和成像参数/技术（ 例如 ，曝光时间，焦距等 ）。相对于前者，在这里描述的方案的活细胞的研究是有限的，以通用的膜和细胞质标记，报告在实时T细胞的APC接口的拓扑变化 （例如， 图2中，视频1的转染， 2）。此外，该协议可以很容易地通过伴随引入/成像进一步记者在内皮（修改，以用于更复杂的分析，例如 ，荧光蛋白标记的MHCI / II和粘附，共刺激和共抑制分子和生物传感器用于信令和生物力学动力学）。然而不幸的是，一般的限制是，淋巴细胞是非常困难的转染。这排除使用方便荧光蛋白的T细胞为活细胞成像（例如，标记，以TCR，PKC-Q 等 ）。 而目前的协议包括通过超抗原（一种广泛使用的模型）激活人体效应/记忆CD4 + Th1型淋巴细胞，更广泛的可能性存在。正如以前证明19,20这种方法很容易适应范围广泛的其他设置，例如其他的 CD4 +亚群和活化的CD8 +淋巴细胞杀伤应答响应。通过从现有的TCR转基因株的淋巴细胞和内皮细胞的分离这种方法也易于适应研究应对特定肽抗原19,20。最后，目前的做法是限制在检查效应/记忆T细胞的。然而，内皮细胞中的基本共刺激分子（CD80 / 86，通常不表达在内皮上）可能允许在该模型中幼稚细胞引发动力学研究的转染。

T淋巴细胞是其特征在于，以有效地识别肽抗原（Ag）材料的能力适应性免疫系统势必主要组织相容性复合体（MHC）的一个分支分子通过它们的T细胞受体（TCR）1。幼稚淋巴细胞组成迁移和扫描的专业抗原呈递细胞"（的APC;例如，树突细胞）的淋巴结内，而记忆/效应T细胞需要能够有效地测量极其广泛外周组织内APC和潜在的靶细胞。 在下面的APC的初始确认同源Ag的分，淋巴细胞逮捕他们的迁移，并开始形成一个专门的亲密细胞间的接口称为"免疫突触"（IS）。持续（即，30-60分钟）IS触点需要以扩增和维持信令2-7。新兴的研究确定了IS内，它是在连续的形成和快速řemodeling离散的亚细胞信号传导的微群 （例如，含有MHC /银TCR，F-肌动蛋白，黏附和信号分子）确定的强度和产生的免疫反应2-7质量。然而，动态信息这一过程和调控机制不完全理解8,9。这在很大程度上源于与APC的表面不规则拓扑和细胞-细胞相互作用平面的控制不良取向，问题的深刻限制所需时空成像相关联的技术挑战接近8-10（Figure1A）。 <img alt="图1" src="/files/ftp_upload/53288/53288fig1.jpg" /> 图1.生理平面成像免疫突触动力电池APC模型。该图说明免疫突触的T细胞和professio之间的传统影像<html>最终APC（A）和T细胞与传统的平面脂双层APC模型（B）相比，这种新型内皮平面APC模式（C）。专职APC提供生理免疫突触，但提供取向不良的细胞-细胞接口（即相对于该最佳的xy成像平面;分辨率〜0.2微米），这显着地损害了空间（Z成象平面分辨率〜1微米）和时间（即由于需要通过所有的z成像平面）成像的分辨率反复扫描。双层车型有平面拓扑结构，提供最佳的时空成像分辨率，而且还高度简化，非生理性和刚性。这种内皮细胞模型结合脂质双层的平面拓​​扑与经典的APC的生理基板于生理环境提供最佳的空间和时间分辨率的成像。M /文件/ ftp_upload / 53288 / 53288fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 以前的工作已部分规避这些障碍通过开发平面衬底的模型（即，脂双层和抗体包被的表面上）提供最佳的时空分辨率（即，经由T细胞活化表面固定到一个单一的计划，是平行于最佳的xy成像平面）11-15日 （图1B）。这些模式促进了重要的见解控制抗原信号的T细胞，包括动态肌动蛋白/ TCR信号微群7,11-14的发现的亚细胞/分子动力学。然而，这种模型被固有地过于简化，以及刚性（排除的三维拓扑特征的发展/研究）（ 图1B）。因此，尚不能确定如何与这样的调查结果PHYsiologic细胞与细胞免疫监视。 虽然仍然充分研究，血管及淋巴管内皮细胞正在成为一个大的（ 即 ，更大数量比所有专业的装甲运兵车，由〜1000倍）的"半专业"的APC 16-18周边室。这些细胞表达MHC-I-，MHC-II-和多种共刺激分子（例如，CD40，LFA3，ICOSL，4-1BB，OX40L，TL1A，PD-L1，但不是CD80和CD86），并在战略上是位于血液组织界面，他们成为专业的定点功能16-18。以往的研究表明，内皮细胞可以有效地重新刺激效应/记忆，但不幼稚，T细胞19-25。因此，血管内皮细胞有可能在周围组织内的适应性免疫反应，如对T细胞的活化，分 ​​化，存储器和公差16,17,26局部影响的效应阶段发挥独特的APC的作用。 CRI的角度讲，当在体外生长的细胞，内皮细胞形成几乎平坦的细胞表面，且很容易转染的（例如，用荧光蛋白记者）。这些特性非常适合拓扑动态过程中细胞间的相互作用19,27的高时空分辨率的成像。因此，内皮细胞可作为生理"平面细胞APC"的模式明显适合于驱动抗原识别和调节反应（ 图1C）19,20的亚细胞/分子重塑机制的研究。 先前建立互补的成像技术（包括内皮细胞的细胞膜和胞浆内的荧光蛋白制造商的转染），用于粘附和跨内皮迁移27期间学习白细胞-内皮相互作用的细节，显示，白细胞由动态主动探测内皮的表面插入的亚微米级的D-回缩，肌动蛋白富圆柱形突起（〜在直径和深度200-1,000纳米）称为invadosome状突起（即，&#39;ILPS&#39;）27,28。这些成像方法已被进一步扩大以及创造的协议采取的内皮APC功能优势，开发的第一个方法为T细胞与内皮细胞免疫突触的高时空分辨率成像报告19,20和进一步说明在此。从这个新的平面细胞APC模型推导出一个核心发现是，T细胞ILPS无论是在推动初期银检测和维持后续信号功能。事实上，多个ILPS（被稳定和应计响应初始钙通量）显示富集TCR和分子暗示的积极信号，例如PKC-Q，ZAP-70，磷酸和HS1的阵列。因此，ILPS似乎代表一个三维生理等效于TCR的信令微集群出现在平面双层模型。这种做法，因此，敏感地揭示/报道分子和建筑（以及隐含的生物力学）动力学没有其他检测。 本文所描述的方法应是有益的桥连专职APC和人工APC的基底模型之间的间隙，以提高我们的询问适应性免疫应答的基本机制的能力。而这里的重点是对CD4 + Th1型效应/记忆细胞的活化，这基本的方法可以容易地修改，以研究各种T细胞类型和AGS，如下面所讨论。

<table><tbody><tr><td>BD Vacutainer stretch latex free tourniquet</td><td>BD Biosciences</td><td>367203</td><td></td></tr><tr><td>BD alcohol swabs</td><td>BD Biosciences</td><td>326895</td><td></td></tr><tr><td>BD Vacutainer Safety-Lok</td><td>BD Biosciences</td><td>367861</td><td>K2 EDTA</td></tr><tr><td>BD Vacutainer Push Button Blood Collection Set</td><td>BD Biosciences</td><td>367335</td><td></td></tr><tr><td>RPMI-1640</td><td>Sigma-Aldrich</td><td>R8758-1L</td><td></td></tr><tr><td>Ficoll-Paque</td><td>Sigma-Aldrich</td><td>GE17-1440-02</td><td>Bring to RT before use</td></tr><tr><td>FCS-Optima</td><td>Atlanta Biologics</td><td>s12450</td><td>Heat inactivated</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Aldrich</td><td>P4458-100ML</td><td></td></tr><tr><td>Trypan blue</td><td>Sigma-Aldrich</td><td>T8154-20ML</td><td></td></tr><tr><td>staphylococcal enterotoxin B</td><td>Toxin Technology</td><td>BT202RED</td><td>Stock solution 1mg/ml in PBS</td></tr><tr><td>toxic shock syndrome toxin 1</td><td>Toxin Technology</td><td>TT606RED</td><td>Stock solution 1mg/ml in PBS</td></tr><tr><td>human IL-15</td><td>R&amp;amp;D Systems</td><td>247-IL-025</td><td>Stock solution 50ug/ml in PBS</td></tr><tr><td>PBS</td><td>Life Technologies</td><td>10010-049</td><td></td></tr><tr><td>Fibronectin</td><td>Life Technologies</td><td>33016-015</td><td>Stock solution 1mg/ml in H20</td></tr><tr><td>HMVEC-d Ad-Dermal MV Endo Cells</td><td>Lonza</td><td>CC-2543</td><td>Other Human Microvascular ECs can be used, i.e. HLMVECs</td></tr><tr><td>EGM-2 MV bullet kit</td><td>Lonza</td><td>CC-3202</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Sigma-Aldrich</td><td>T-4174</td><td>Stock solution 10x, dilute in PBS</td></tr><tr><td>amaxa-HMVEC-L Nucleofector Kit</td><td>Lonza</td><td>vpb1003</td><td>Required Kit for step 4</td></tr><tr><td>IFN-g</td><td>Sigma-Aldrich</td><td>I3265</td><td>Stock solution 1mg/ml in H20</td></tr><tr><td>TNF-alpha 10ug, human</td><td>Life Technologies</td><td>PHC3015</td><td>Stock solution 1mg/ml in H20</td></tr><tr><td>phenol Red-free HBSS</td><td>Life Technologies</td><td>14175-103</td><td></td></tr><tr><td>Hepes</td><td>Fisher Scientific</td><td>BP299-100</td><td></td></tr><tr><td>Calcium Chloride</td><td>Sigma-Aldrich</td><td>C1016-100G</td><td>Stock solution 1M in H20</td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>208337</td><td>Stock solution 1M in H20</td></tr><tr><td>Human Serum albumin</td><td>Sigma-Aldrich</td><td>A6909-10ml</td><td></td></tr><tr><td>Immersol 518 F fluorescence free Immersion oil</td><td>Fisher Scientific</td><td>12-624-66A</td><td></td></tr><tr><td>Fura-2 AM 20x50ug</td><td>Life Technologies</td><td>F1221</td><td>Stock solution 1mM in DMSO</td></tr><tr><td>pEYFP-Mem (Mem-YFP)</td><td>Clontech</td><td>6917-1</td><td></td></tr><tr><td>pDsRed-Monomer (Soluble Cytoplasmic DsRed)</td><td>Clontech</td><td>632466</td><td></td></tr><tr><td>pDsRed-Monomer Membrane (Mem-DsRed)</td><td>Clontech</td><td>632512</td><td></td></tr><tr><td>pEGFP-Actin</td><td>Clontech</td><td>6116-1</td><td></td></tr><tr><td>Alexa Fluor 488 Phalloidin</td><td>Life Technologies</td><td>A12379</td><td></td></tr><tr><td>Formaldehyde solution 37%</td><td>Fisher Scientific</td><td>BP531-500</td><td>Toxic, use fumehood</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100-5ML</td><td></td></tr><tr><td>&amp;nbsp;Falcon 15mL Conical Centrifuge Tubes</td><td>Fisher Scientific</td><td>14-959-70C</td><td></td></tr><tr><td>Falcon 50mL Conical Centrifuge Tubes</td><td>Fisher Scientific</td><td>14-959-49A&amp;nbsp;</td><td></td></tr><tr><td>Falcon Tissue Culture Treated Flasks T25</td><td>Fisher Scientific</td><td>10-126-9</td><td></td></tr><tr><td>&amp;nbsp;Falcon Tissue Culture Treated Flasks T75</td><td>Fisher Scientific</td><td>&amp;nbsp; 13-680-65</td><td></td></tr><tr><td>&amp;nbsp;Corning Cell Culture Treated T175</td><td>Fisher Scientific</td><td>10-126-61&amp;nbsp;</td><td></td></tr><tr><td>Glass coverslips&amp;nbsp;</td><td>Fisher Scientific</td><td>12-545-85&amp;nbsp;</td><td>12 mm diameter</td></tr><tr><td>&amp;nbsp;Falcon Tissue Culture Plates 24-well</td><td>Fisher Scientific</td><td>08-772-1</td><td></td></tr><tr><td>Delta-T plates</td><td>Bioptechs</td><td>04200415B</td><td></td></tr><tr><td>Wheaton Disposable Pasteur Pipets</td><td>Fisher Scientific</td><td>13-678-8D</td><td></td></tr><tr><td>1.5 ml Eppendorf tube&amp;nbsp;</td><td>Fisher Scientific</td><td>05-402-25</td><td></td></tr><tr><td>&amp;nbsp;ICAM1 mouse anti-human</td><td>BD Biosciences</td><td>555509</td><td></td></tr><tr><td>HS1 mouse anti-human</td><td>BD Biosciences</td><td>610541</td><td></td></tr><tr><td>Anti-Human CD11a (LFA-1alpha) Purified</td><td>ebioscience</td><td>BMS102</td><td></td></tr><tr><td>Anti-Human CD3 Alexa Fluor&amp;reg; 488</td><td>ebioscience</td><td>53-0037-41</td><td></td></tr><tr><td>Anti-MHC Class II antibody&amp;nbsp;</td><td>Abcam</td><td>ab55152</td><td></td></tr><tr><td>Anti-Talin 1 antibody</td><td>Abcam</td><td>ab71333</td><td></td></tr><tr><td>Anti-PKC theta antibody&amp;nbsp;</td><td>Abcam</td><td>ab109481</td><td></td></tr><tr><td>phosphotyrosine (4G10 Platinum)</td><td>Millipore</td><td>50-171-463</td><td></td></tr><tr><td>Nucleofector II</td><td>Amaxa Biosystems</td><td></td><td>Required electroporator for step 4</td></tr><tr><td>Zeiss Axiovert</td><td>Carl Zeiss MicroImaging</td><td></td><td></td></tr><tr><td>Zeiss LSM510&amp;nbsp;</td><td>Carl Zeiss MicroImaging</td><td></td><td></td></tr><tr><td>Zeiss Axiovison Software</td><td>Carl Zeiss MicroImaging</td><td></td><td></td></tr><tr><td>NU-425 (Series 60) Biological Safety Cabinet</td><td>NuAIRE</td><td>Nu-425-600</td><td></td></tr><tr><td>&amp;nbsp;Forma STRCYCLE 37 &amp;deg;C, 5% CO2 Cell culture Incubator</td><td>Fisher Scientific</td><td>202370</td><td></td></tr><tr><td>Centrifuge 5810</td><td>Eppendorf</td><td>EW-02570-02</td><td></td></tr><tr><td>Hemocytometer</td><td>Sigma-Aldrich</td><td>&amp;nbsp;Z359629</td><td>Bright-Line Hemocytometer</td></tr><tr><td>Isotemp Waterbath model 202</td><td>Fisher Scientific</td><td>15-462-2</td><td></td></tr></tbody></table>

an endothelial planar cell model for imaging immunological synapse dynamics

适应性免疫是通过T细胞和抗原呈递细胞（&#39;的APC&#39;）之间动态相互作用调节被称为"免疫突触&#39;。在这些贴心的细胞与细胞间的接口MHC /银TCR，F-肌动蛋白，粘连分离亚细胞簇和信号分子形成和重塑迅速。这些动力被认为是双方的效率和发展的免疫应答的质量的关键因素，因此，保护​​与病理性免疫。免疫突触生理装甲运兵车的当前的理解是可获得的成像分辨率不足的限制。尽管人造材料模型 （例如，平面脂质双层）提供了出色的分辨率和一直非常有价值的工具，它们是固有的非生理性和过于简单。血管和淋巴管内皮细胞已经成​​为"半职业的一个重要的外周组织（或基质）隔人装甲运兵车"。这些装甲运兵车（其中表达最专业的装甲运兵车的分子机器）都形成几乎平坦的细胞表面的独特的功能，且很容易转染的（例如，用荧光蛋白记者）。本文将描述一个基本的方法来实现的内皮细胞作为一种新颖的和生理&#39;平面蜂窝APC模式"为改进的成像和基本的抗原性信令过程的询问。

使用内皮细胞和结合生理复杂性和专职APC变形的平面脂双层模型的分辨率优点的新型成像方法被开发（图1）。图2提供了典型的迁移，钙流和拓扑动力学与此观察的例子做法。在内皮上没有凹陷，超抗原特异性的 CD4 + Th1细胞淋巴细胞迅速蔓延，极化和横向上内皮表面（图2A）迁移，而熔合钙（未示出）。在〜5-10分钟，这些细胞开始穿过内皮细胞轮回。在超抗原装载的存在到内皮，淋巴细胞对称地以&#39;煎蛋&#39;拓扑扩散，应启动持续（即，30〜60分钟）的细胞内钙通量（ 图2B;电影1），在此期间它们展现很少或没有迁移（ 图2C）。 <img alt="图2" src="/files/ftp_upload/53288/53288fig2.jpg" /> T细胞的活化和迁移和内皮装甲运兵车的免疫突触的拓扑结构。（A）面板图2.活细胞分析显示T细胞迁移对血管内皮细胞在没有凹陷时序成像的一个例子。上图显示了DIC图像。迁移T细胞的初始位置由红色虚线表示。下部面板示出了由迁移T细胞的形成在内皮内陷。箭头表示形成膜-YFP荧光形成为T细胞产生&#39;invadosome状突起&#39;（ILPS）对内皮表面的APC（见D）的结果瞬态环（〜0.5-1微米直径）。 （b）示出了类似的系列中SAG-加载 ​​内皮（左的存在的例子;也参见相应的电影1）和钙含量（右）的动态变化的定量跟踪。箭头指示初始ILPS形成，随着钙流（i）所述初始关联和多ILPS随后稳定以高钙流（ⅱ）相关。 （C）的实验如在A和B经受细胞跟踪分析。 DIC影像超过10分钟（红色痕迹）期间左秀代表T细胞的迁移路径。在右边的图显示在没有和凹陷存在计算的迁移速度。 （D）免疫突触的拓扑结构（从19修改）的动态分析。原理图（一）说明了一个敏感的内皮细胞/ APC"拓扑报告系统"组成的膜有针对性的-YFP和可溶性胞浆RFP的。肌动蛋白 - 介导的ILP伸入内皮细胞表面生成筒状的内陷（一种类型的蜂窝尺打印，称为&#39;PODO印刷&#39;）克艾尔文引起急剧弯曲膜，其表现为膜-YFP荧光环（ 即 ，观看恩面对气缸的壁）。细胞质位移和排斥，在这些位点出现胞浆RFP的黑眼圈。 DIC和（ii）中示出这样的分析在超抗原的存在的一个例子的荧光图像（参见对应电影2）。一个单一的初始PODO打印/ ILP发展到一个数组中的&gt; 20稳定ILPS超过15分钟。在15分钟（ⅲ）蓝色框图示由此膜-YFP的环带排除胞质RFP的一区域重叠的单个PODO打印/ ILP的高分辨率图。虚线（ⅳ）表示荧光强度的定量线扫描分析。比例尺= 5微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53288/53288fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 正如前面所建立的，淋巴细胞ACTIV伊利探针内皮通过延伸肌动蛋白和HS1（一皮层蛋白同源物）的表面富集的和WASp-和src依赖性突起称为"invadosome状突起; （ILPS;每个〜0.5-1微米的深度，直径）27,28。膜靶向荧光蛋白（FP）的转染已被证明在细胞 - 细胞表面亚细胞拓扑动力学提供高度敏感的记者。以这种方式，它已被证明荧光膜-FP的该亮环是指示膜中的T细胞 - 内皮相互作用的T细胞ILPS突起的结果的弯曲。这显示在这里，并在以往的研究19,20，而这小簇的ILPS形式和营业额迅速（ 即 10-30秒〜寿命）T细胞对血管内皮细胞在没有凹陷的横向迁移过程中（ 图2A ），他们成为高度稳定（ 即中，约30分钟的寿命）和积累成密集阵列的存在凹陷（ 图2B）。此外，ILPS和钙引发动力学分析（"补偿时间"）显示，ILPS先钙信号，这表明他们在MHC-II /银识别过程（ 图2Bii）援助。此外，稳定ILPS阵列形成具有最大的钙通量（ 图2Bii）相称。有观点认为，膜荧光环对应于T细胞突起驾驶离散内陷景点内皮装甲运兵车，是由这些环在所有情况下相关的空间和动力学流离失所/排除细胞质的区域（所报告的可溶性红色荧光蛋白论证确定; 图2D和电影2）。 图3提供固定端点聚焦成像研究的例子。经过共培养的凹陷，国际空间站的T细胞和内皮装甲运兵车之间形成的存在，10分钟由固定进行成像，我mmuno荧光染色和共聚焦显微镜。这些研究提供了重要的分子的亚细胞分布的动态分析和信令活动范围内的IS尤其涉及ILPS。具体地，细胞骨架/细胞骨架调节器（肌动蛋白，HS1; 图3A），抗原呈递/识别（CD3 / MHC-II; 图3B）附着力（ICAM-1，LFA-1，踝蛋白; 图3C），和抗原信令（PKC -Q; 图3D）被视为要共富含PODO印痕/ ILP。的共焦串行部分的z栈数字重建提供了互补的证据与图2D所示的T细胞-内皮内皮IS具有离散的3维结构由T细胞ILP在APC的表面突出打断（图3Aii， 次 ，三，四）。 （ 图3Civ）。 <img alt="图3" src="/文件/ ftp_upload / 53288 / 53288fig3.jpg"/&gt; 在T细胞内皮细胞免疫突触分子动力学分布图3.固定样本分析。淋巴细胞孵育上超抗原加载的内皮30分钟，固定并染色。在显示面板内皮细胞进行预转染以表达膜YFP或-DsRed。 （A）的代表性的成像显示肌动蛋白（i）和HS1（一个皮层蛋白同源物;ⅱ）共富集在免疫突触中的微簇，随着膜-YFP的内皮荧光环（ 即 ，&#39;中心相关联的周边PODO版画"; 见图2D），确认T细胞ILPS负责外观PODO-打印。的侧面图（ⅱ。90°投影）图示ILPS表示在 z成像平面（从19修改）的三维凸起。 （B）T细胞TCR（i）和endoth代表富集elial的MHC-II（ii）所述免疫突触中，显示离散的亚细胞划分到ILP / PODO印痕。 （C）（Ⅰ）代表PODO印和ILP，共同致富ICAM-1和LFA-1分别IS的。 （i）表示的本IS的共聚焦部分的3-D数字重建旋转60°（上图）和90°（正交视图;下面板）。 （二）显示IS 恩面对的放大图。 （ⅲ）显示盒装区域（ii）中（从19修改）的正交截面图。 （四）代表共同致富塔林（一integtin /肌动蛋白连接蛋白）和肌动蛋白的IS ILPS。 （四）代表共同致富的TCR信号分子PKC-Q与肌动蛋白的IS ILPS。每个面板显示的连接面视图（左下部分）和两个不同的正交视图（90°投影）。最左边和右边的面板用的都是自己的，各相邻板的重复，采用了彩虹fluorescenc预测Ë烈度表（从19修改）。比例尺= 5微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53288/53288fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="电影1" src="/files/ftp_upload/53288/53288movie1.jpg" /> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53288/53288movie1.mov">电影1:T细胞钙信号活细胞动态成 ​​像和免疫突触拓扑</a>的Fura2加载的 CD4 + Th1细胞淋巴细胞与内皮表达膜-YFP和装载有凹陷， 对应于图2B的交互定时活细胞成像。左图显示了细胞内钙水平（彩虹:冷色调，低钙，温暖的色调高钙）的T细胞。内皮APC表面的右面板中显示膜YFP荧光。淋巴细胞被认为以引发扩频和ILP形成（如通过荧光环在MEM-YFP的信道看到的;也就是，&#39;PODO印痕&#39;; 见图2BI），接着诱导持续（30-60分钟）的细胞内钙通量的，这发生在形成ILPS / PODO印痕类似于TCR /肌动蛋白的外围阵列相称的"信号微集群"的是出现在平面双层APC模型（ 见图2Bii）。帧之间的时间间隔为20秒。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53288/Movie1_finale_02_18_15.mov" target="_blank">请点击此处观看该视频。</a> <img alt="电影2" src="/files/ftp_upload/53288/53288movie2.jpg" /> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53288/53288movie2.mov">电影2:免疫突触拓扑六活细胞动态成 ​​像</a>与内皮交互的荧光记者在内皮的APCs。的CD4 + Th1细胞淋巴细胞定时活细胞成像（DIC，左）共表达膜-YFP（绿色）和可溶性细胞质DsRed的（红色）和装载有凹陷，对应于图2D。右图显示的DIC，细胞膜和细胞质的信号合并。细胞膜和细胞质荧光的组合信号灵敏报告的形成多与重塑〜0.2-1微米级的表面内陷（PODO印痕）所造成的阵列T细胞与ILP突起探测。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53288/Movie2_finale_02_18_15.mov" target="_blank">请点击此处观看该视频。</a>

Watch this Scientific Journal Video about An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics at JoVE.com

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

成像免疫突触动力学内皮平面细胞模型

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological ...

an-endothelial-planar-cell-model-for-imaging-immunological-synapse

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.9K 次观看.  Harvard Medical School - BIDMC. Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

视频: An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

艾滋病毒的成像1信封引起的病毒学突触信号合成脂双层

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

科研

免疫与感染

Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions between lymphocytes and antigen presenting cells, we use Ni2+-chelating lipids to anchor recombinant cell adhesion and MHC proteins to the upper leaflet of a bilayer with poly-histidine tags. Planar bilayers are generated by preparing lipid, treating the glass surfaces where the bilayer will form, and then forming the bilayer in a specialized chamber containing a flow-cell where the lymphocytes will be added.  Then, bilayers are charged with Ni and his-tagged recombinant proteins are added.  Finally,  lymphocytes are injected into the flow cell and TIRF microscopy can be used to image synapse formation and the mechanisms that control T cell locomotion, sites of receptor sorting,  and sites of receptor degradation.

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse. Here, we show methods for anchoring cell adhesion proteins known to modulate synapse formation to the upper leaflet of the lipid bilyer and visualize synapse formation using TIRF microscopy.

形成免疫突触和Kinapse研究的支持平面双层膜

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions ...

生物学

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion (STED) Nanoscopy

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic actin reorganization is critical to the function of NK cells and the formation of the IS. Imaging of F-actin at the synapse has traditionally utilized confocal microscopy, however the diffraction limit of light restricts resolution of fluorescence microscopy, including confocal, to approximately 200 nm. Recent advances in imaging technology have enabled the development of subdiffraction limited super-resolution imaging. In order to visualize F-actin architecture at the IS we recapitulate the NK cell cytotoxic synapse by adhering NK cells to activating receptor on glass. We then image proteins of interest using two-color stimulated emission depletion microscopy (STED). This results in &#60;80 nm resolution at the synapse. Herein we describe the steps of sample preparation and the acquisition of images using dual color STED nanoscopy to visualize F-actin at the NK IS. We also illustrate optimization of sample acquisition using Leica SP8 software and time-gated STED. Finally, we utilize Huygens software for post-processing deconvolution of images.

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion STED Nanoscopy

Here we illustrate the protocol for imaging by two-color STED nanoscopy the cytotoxic immune synapse of NK cells recapitulated on glass. Using this method we obtain sub-100 nm resolution of synapse proteins and the cytoskeleton.

免疫突触由双色时间选通受激发射损耗（STED）纳米显微可视化

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

内皮细胞，细胞连接实时成像过程中中性粒细胞轮回在生理流动

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

的自然杀伤细胞免疫突触上的玻璃支承平面脂双层超分辨率成像

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to study the immune synapse. Since these methods require manual image acquisition and time-consuming quantification, the imaging of rare events is challenging. Here, we describe a workflow that enables the morphological analysis of tens of thousands of cells. Immune synapses are induced between primary human T cells in pan-leukocyte preparations and Staphylococcus aureus enterotoxin B (SEB)-loaded Raji cells as APCs. Image acquisition is performed with imaging flow cytometry, also called In-Flow microscopy, which combines features of a flow cytometer and a fluorescence microscope. A complete gating strategy for identifying T cell/APC couples and analyzing the immune synapses is provided. As this workflow allows the analysis of immune synapses in unpurified pan-leukocyte preparations and hence requires only a small volume of blood (i.e., 1 mL), it can be applied to samples from patients. Importantly, several samples can be prepared, measured, and analyzed in parallel.

Here, we describe a complete workflow for the qualitative and quantitative analysis of immune synapses between primary human T cells and antigen-presenting cells. The method is based on imaging flow cytometry, which allows the acquisition and evaluation of several thousand cell images within a relatively short period of time.

影像性流式细胞仪对人体免疫突触的定性和定量分析

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

人外周血和淋巴组织中原发性 T 细胞突触界面的评价

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. Transcytosis across the BBB regulates the delivery of hormones, metabolites, and therapeutic antibodies to the brain parenchyma. Here, we present a protocol that combines immunofluorescence of free-floating sections with laser scanning confocal microscopy and image analysis to visualize subcellular organelles within endothelial cells at the BBB. Combining this data-set with 3D image analysis software allows for the semi-automated segmentation and quantification of capillary volume and surface area, as well as the number and intensity of intracellular organelles at the BBB. The detection of mouse endogenous immunoglobulin (IgG) within intracellular vesicles and their quantification at the BBB is used to illustrate the method. This protocol can potentially be applied to the investigation of the mechanisms controlling BBB transcytosis of different molecules in vivo.

Here we present a microscopy-based protocol for high-resolution imaging and a three-dimensional reconstruction of the mouse neurovascular unit and blood-brain barrier using brain free-floating sections. This method allows for the visualization, analysis, and quantification of intracellular organelles at the BBB.

血脑屏障的高分辨率共聚焦成像: 成像、3D 重建和 Transcytosis 的量化

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. ...

神经科学

Imaging the Human Immunological Synapse

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) and an effector helper T lymphocyte (Th) cell, and to record the images corresponding to the first stages of the IS formation and the subsequent trafficking events (occurring both in the APC and in the Th cell). These events will eventually lead to polarized secretion at the IS. In this protocol, Jurkat cells challenged with Staphylococcus enterotoxin E (SEE)-pulsed Raji cells as a cell synapse model was used, because of the closeness of this experimental system to the biological reality (Th cell-APC synaptic conjugates). The approach presented here involves cell-to-cell conjugation, time-lapse acquisition, wide-field fluorescence microscopy (WFFM) followed by image processing (post-acquisition deconvolution). This improves the signal-to-noise ratio (SNR) of the images, enhances the temporal resolution, allows the synchronized acquisition of several fluorochromes in emerging synaptic conjugates and decreases fluorescence bleaching. In addition, the protocol is well matched with the end point cell fixation protocols (paraformaldehyde, acetone or methanol), which would allow further immunofluorescence staining and analyses. This protocol is also compatible with laser scanning confocal microscopy (LSCM) and other state-of-the-art microscopy techniques. As a main caveat, only those T cell-APC boundaries (called IS interfaces) that were at the right 90&#176; angle to the focus plane along the Z-axis could be properly imaged and analyzed. Other experimental models exist that simplify imaging in the Z dimension and the following image analyses, but these approaches do not emulate the complex, irregular surface of an APC, and may promote non-physiological interactions in the IS. Thus, the experimental approach used here is suitable to reproduce and to confront some biological complexities occurring at the IS.

This protocol images both the immunological synapse formation and the subsequent polarized secretory traffic towards the immunological synapse. Cellular conjugates were formed between a superantigen-pulsed Raji cell (acting as an antigen-presenting cell) and a Jurkat clone (acting as an effector helper T lymphocyte).

成像人类免疫学突触

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) ...

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. Although studying these cells in isolation in vitro or together ex vivo provides useful physiological information; salient features of neural cell physiology will be missed in such contexts. Therefore, there is a need for studying neural cells in their native in vivo environment. The protocol detailed here describes repetitive in vivo two-photon imaging of neural cells in the rodent cortex as a tool to visualize and study specific cells over extended periods of time from hours to months. We describe in detail the use of the grossly stable brain vasculature as a coarse map or fluorescently labeled dendrites as a fine map of select brain regions of interest. Using these maps as a visual key, we show how neural cells can be precisely relocated for subsequent repetitive in vivo imaging. Using examples of in vivo imaging of fluorescently-labeled microglia, neurons, and NG2+ cells, this protocol demonstrates the ability of this technique to allow repetitive visualization of cellular dynamics in the same brain location over extended time periods, that can further aid in understanding the structural and functional responses of these cells in normal physiology or following pathological insults. Where necessary, this approach can be coupled to functional imaging of neural cells, e.g., with calcium imaging. This approach is especially a powerful technique to visualize the physical interaction between different cell types of the CNS in vivo when genetic mouse models or specific dyes with distinct fluorescent tags to label the cells of interest are available.

This protocol describes a chronic cranial window implantation technique that can be used for longitudinal imaging of neuro-glio-vascular structures, interactions, and function in both healthy and diseased conditions. It serves as a complementary alternative to the transcranial imaging approach that, while often preferred, possesses some critical limitations.

精确的大脑映射，在小鼠中执行神经-免疫动力学的重复体内振动成像

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. ...