作者感谢索菲亚炸的技术援助。 MBS感谢以下机构为他们的研究支持:以色列科学基金会补助no.1659/08，971/08，1503/08和491/10，没有补助的健康与科学部。 3-4114和3-6540，透过地产已故的亚历山大Smidoda的，以色列癌症协会和Taubenblatt家庭基金会为生物医药的卓越补助。

1。 T细胞转染<ol><li>准备激活Amaxa Nucleofector溶液混合套件的"补充说明"解决Nucleofector解决方案。激活的解决方案可以被存储在4℃下为不超过三个月。 </li><li>成长Jurkat T细胞，在培养基中[10％胎儿小牛血清（FCS），1％青霉素 - 链霉素溶液，2mM L-谷氨酰胺的RPMI。理想情况下，应该使用细胞解冻后1-2周。使用对数生长期的细胞培养，转染效率高和细胞生存的关键。或者，稍作修改，该协议可以使用的主要的T细胞，如前面所述。应该强调的是，作为主T细胞的激活过程中的持续时间长于Jurkat T细胞（〜30分钟），显微镜载物台上安装孵化体系必须被用来维持CO 2，湿度水平和温度在整个激活过程。 </li><li>收集5个×10 6对数生长期的T细胞转染每到15毫升管。 </li><li>离心7分钟，在400×g下在室温下的细胞。 </li><li>在离心过程中，准备一个6孔板。填补以及为每个转染在37℃下用5毫升培养基，预孵育</li><li>收集管从离心机中，并弃去上清液。在1毫升的RPMI重悬细胞沉淀。 </li><li>离心7分钟，在400×g下在室温下的细胞。 </li><li>在离心过程中，准备转染的解决方案。在一个孔的96孔板中，加入100μl混合激活Amaxa Nucleofector在步骤1中制备的溶液与5微克的质粒DNA的编码荧光标记的蛋白的权益。理想情况下，DNA浓度应大于1微克/毫升，以避免稀释的转染溶液。 </li><li>轻柔吹打拌匀。 </li><li>细胞离心后，小心地取出所有的上清，而不是Disturbing沉淀。 </li><li>移液器的DNA /转方案的两倍。 </li><li>加入沉淀DNA /转染溶液。 </li><li>细胞转移到Amaxa比色皿。 </li><li>试管插入到Amaxa Nucleofector设备。 </li><li>使用H-10 Amaxa转设置电（为JURKAT）的T-23（主T细胞）。 </li><li>从培养箱中取出6孔板。 </li><li>电穿孔后，小心地用细胞悬浮液将上清转移到6孔板，使用巴斯德吸管。一种粒料组成的细胞碎片可能形成。避免转移的沉淀。 </li><li>孵育细胞24小时，在37°C。 </li><li> 2.5 ml的细胞悬液转移到一个新的井每口井。向每孔中加入2.5毫升温暖的培养基中（在步骤2中所述）。 </li><li> 72小时后，细胞转移到一个无菌管和离心7分钟，在400×g下在室温下。弃上清，取代它的Wi个培养基中补充了哺乳动物细胞选择适当的抗生素所用的质粒（适用的浓度适合于所使用的抗生素，我们用G418和/或潮霉素浓度为1.3毫克/毫升和/或0.2毫克/毫升，分别）。在一个新鲜的以及文化的细胞悬液。另外，细胞可被转染瞬时成像转染后48小时。要准备瞬时转染细胞，共同转染细胞与两个质粒同时在第8步，继续正常步骤20，然后直接跳到步骤23。请注意，在这种情况下，细胞应立即使用，一般其荧光强度异构不一定高。 </li><li>辅以适当的选择抗生素的培养基培养细胞。分裂细胞，并补充以补充抗生素的培养基中，根据需要。 </li><li> 2周后，使用流式细胞仪（流感验证成功转染orescence激活细胞分选），如下所示: </li><li>收集10 6转染的细胞，10 6模拟转染的细胞（阴性对照），和10 6个细胞是已知的稳定表达荧光标记的蛋白。如果与一个以上的荧光蛋白（见步骤27）转染的细胞，使用多个单独使用的标签，作为阳性对照的细胞系。 </li><li>离心7分钟，在400×g下在室温下的细胞。 </li><li>弃上清。将细胞重新悬浮于500μlFACS缓冲液（PBS W / O型的Ca 2 +和Mg 2 +，5％FCS，0.05％叠氮化钠）。 </li><li>使用一个分析的流式细胞仪来验证细胞荧光。比较转染的细胞的荧光的阴性和阳性对照。 </li><li>转染的细胞与一个额外的，荧光标记的蛋白质，重复步骤3-26，使用转染的细胞。 </li><li>显示高水平的荧光的细胞应该被使用。最理想的情况是，至少50％的在培养的细胞应该是高荧光。通过流式细胞仪的细胞分选，可以增加细胞荧光。 </li></ol>任何额外的标记蛋白质，必须融合到不同的荧光基团和不同的选择抗生素质粒编码进行编码。与一个以上的质粒转染的细胞的选择培养基中应补充合适的抗生素。 2。 T细胞传播的含量制备<ol><li>使用实验室泰克II德国玻璃罩系统4玻片。确保不意外划伤腔的内表面， 例如用枪头，准备或使用过程中。 </li><li>准备玻片制备溶液50毫升:12米（37％），盐酸4.6毫升，加入38.5毫升100％的乙醇和6.9毫升二次蒸馏水。 </li><li>填写每个室的幻灯片，用500μl玻片制备解决方案。 </li><li>在室温下孵育10分钟。 </li><li>吸室，它们完全干燥。 </li><li>孵育1小时45°C，直到完全干燥裸露的幻灯片。 </li><li>准备双蒸水稀释的0.1％的聚-L-赖氨酸（Sigma-Aldrich公司）0.01％聚-L-赖氨酸溶液。 </li><li>将500微升每个室。 </li><li>孵育15分钟，在室温下滑动。 </li><li>吸室，它们完全干燥。 </li><li>孵育3小时，在45°C至完全干燥。 </li><li>包被的载玻片，可以存储在室温下干燥数月。 </li><li>准备激活抗体溶液。使用抗CD3抗体:要么HIT3a（BD Pharmingen公司，＃555337）或UCHT1（BD Pharmingen公司，＃555330），10微克/毫升于400μlPBS中，每个腔室。这个解决方案应该准备前不久幻灯片准备。 </li><li>填充每个腔室的活化的抗体溶液，用400μl。 </li><li>孵育2小时，在37℃，或优选的滑动，在4℃下过夜</li><li>删除激活抗体溶胶ution立即洗室300微升PBS两次。从这一点来说，确保商会保持充满缓冲。 </li><li>在4℃下存储的PBS填充的滑动我们建议使用准备幻灯片商会在1周内。 </li></ol> 3。 T细胞的激活与成像<ol><li>准备成像缓冲无酚红] [1.25毫升HEPES（1米），10毫升FCS RPMI，38.75毫升。成像缓冲器可以存储在4℃后，过滤。 </li><li>激活蔡司LSM 510元显微镜。显微镜加热的安装架预热至37°C。选择"专家模式"。 </li><li>点击"获取"选项卡，并打开以下标签:激光显微镜;配置;扫描时间序列。 </li><li>激活所需的激光器。对于激发的MCFP和mYFP设置氩气/ 2激光5分钟，然后到"待机"为"开"。 </li><li>如果你已经执行了这个活细胞成像分析之前，使用相同的荧光团，打开LSM文件，点击"重用"我CON，并继续从第9步。否则，继续从第6步。 </li><li>定义使用过滤器，适用于所使用的荧光蛋白的激发和发射的信道。 </li><li>在"扫描控制"窗口中，选择的Z堆栈选项。 </li><li>输入下面的扫描参数:片数:5;间隔:0.5微米;当前切片:3。 </li><li>准备在显微镜附近一个数字 Accublock 干浴 LABNET在37°C。 </li><li>暖的摄像缓冲液到37℃。 </li><li>准备吸的PBS，取而代之的是用600微升的温暖成像缓冲存储的幻灯片。 </li><li>孵育的幻灯片，在37°C。保持在孵化滑动，直到第13步中，至少15分钟。 </li><li>收集5×10 5转染的T细胞。 </li><li>在400×g离心2分钟的细胞。 </li><li>弃上清。 </li><li> 1毫升温成像缓冲液中的悬浮细胞，并将它们转移到1.5毫升的Eppendorf管。 </li><li>放置吨EST管上的的数字Accublock干浴。 </li><li>从孵化器中加热的幻灯片，把它安装在温暖显微镜安装架。 </li><li>点击"VIS"图标。 </li><li>混合细胞悬液，除去1-2微升。 </li><li>的移液管的尖端插入到成像缓冲器的底部上方的滑动。避免刮伤幻灯片。 </li><li>种子细胞对客观的光圈。 </li><li>视觉跟踪荧光细胞立即开始下降。选择一个单元格，显示在两个通道中的高荧光。 </li><li>前的细胞到达表面的滑动，激活的LSM模式下。 </li><li>切换只有一个激发激光。选择激光，你有DIC通道。 </li><li>设置缩放1。点击"快速XY"图标。点击"停止"图标。使用"裁剪"工具，选择细胞中心的投资回报率。 </li><li>设置缩放至3。点击"快速XY"图标，然后手动将焦点设置到最佳观看细胞。点击"停止"ICO名词</li><li>切换所需的所有激励激光器。 </li><li>开始记录点击的"StartT"图标。图片的单元格的扩展处理的全部（通常为5分钟）。完成后，单击"停止"图标。 </li><li>保存该文件。 </li><li>为了获得更多的细胞，点击"VIS"图标。如果存在还没有接触到的幻灯片的底部的细胞，从步骤23继续。避免成像细胞已经开始蔓延过程。否则，请继续步骤32。 </li><li>如果观察到的幻灯片区域主要是缺乏的细胞，添加另一个细胞（继续从第20步）等分。否则，继续执行步骤33。 </li><li>为了获得额外的电池，移动幻灯片，使该地区上述目标的光圈是缺乏的细胞。 </li><li>添加另一个细胞（和继续从第20步）等分。 </li><li>执行所有所需的成像后，打开保存的LSM文件。 </li><li>点击"图库"，"时间+ Z"和"子集"。 </li><li>选择relevant为时间范围和Z堆栈。 </li><li>保存新的文件。 </li><li>所有影像文件执行步骤35-38。 </li></ol> 4。成像数据分析<ol><li>打开LSM文件使用Imaris里软件（位平面AG，瑞士苏黎世）。 </li><li>点击"超越"。点击"图像处理"菜单，并选择"交换时间和Z"。这将有助于在正确的裁剪的图像。 </li><li>点击"编辑"工具栏，在下拉菜单中选择"剪裁3D"选项。裁剪图像只包括区域周围的细胞。我们建议种植细胞集中在了300×300像素的正方形。需要注意的是细胞的形状和位置可以随时间变化。观察的t轴线，确保细胞在所有时间点的裁剪区域内。 </li><li>点击"图像处理"菜单，并选择"交换时间和Z"恢复原来的时空分离的图像。 </li><li>按"COLOC的"主工具栏上的按钮。选择"生成时间德第COLOC Imaris里会自动计算每个信道在各时间点的强度阈值。 </li><li>共存分析的结果选择"通道统计"。点击"导出"，导出数据。 </li><li>重复步骤1到6，每个成像单元。我们建议在这种方式中，至少50个细胞进行分析。 </li><li>计算平均皮尔逊的共存系数及其标准的错误或偏差。 </li></ol>

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我们没有利益冲突的披露。

多种细胞过程的调节和功能依赖于蛋白质 - 蛋白质相互作用的形成和终止。荧光标记的蛋白质在活细胞显微成像可以实时跟踪。标签的蛋白的共定位，能否提供一个直接或间接的蛋白质之间的相互作用，可以用来加强通过生物化学的方法，如免疫沉淀获得的研究结果。与生化方法不同的是，活细胞成像可以观察这些蛋白间的相互作用空间，随着时间的推移，促进发生的生理过程的监测和检测的蛋?...

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagent/Material&lt;/strong&gt;</td></tr><tr><td>Amaxa human T Cell nucleofector kit</td><td>Lonza</td><td>VCA-1002</td><td></td></tr><tr><td>Anti CD3 (UCHT1 clone) </td><td>BioLegend</td><td>300432</td><td></td></tr><tr><td>Falcon FACS Tubes</td><td>Becton Dickinson</td><td>352058</td><td></td></tr><tr><td>FCS</td><td>HyClone</td><td>SV30160.03</td><td></td></tr><tr><td>G418</td><td>Calbiochem</td><td>345810</td><td></td></tr><tr><td>German coverglass system 4 chamber slides </td><td>Lab-Tek II</td><td>155382</td><td></td></tr><tr><td>HCl</td><td>Bio Lab</td><td>8410501</td><td></td></tr><tr><td>Hepes </td><td>Biological Industries</td><td>03-025-1B</td><td></td></tr><tr><td>Hygomycin</td><td>Enzo</td><td>ALX-380-306</td><td></td></tr><tr><td>L-glutamine </td><td>Sigma</td><td>G7513</td><td></td></tr><tr><td>PBS (10X)</td><td>Sigma</td><td>D1408</td><td></td></tr><tr><td>Penicillin-Streptomycin </td><td>Sigma</td><td>P0781</td><td></td></tr><tr><td>Poly-L-lysine 0.1 % (w/v) in H2O</td><td>Sigma</td><td>P8920</td><td></td></tr><tr><td>RPMI</td><td>Sigma</td><td>R87589</td><td></td></tr><tr><td>Sodium azide</td><td>Sigma</td><td>S2002</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt; </td></tr><tr><td>Accublock digital dry bath</td><td>Labnet</td><td>D1105A</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>Centrifuge 5810 R</td><td></td></tr><tr><td>Confocal microscope</td><td>Zeiss</td><td>LSM 510 Meta</td><td></td></tr><tr><td>Heatable mounting frame - Heating Insert P S </td><td>PeCon</td><td>130-800-031</td><td></td></tr><tr><td>TempModule S (required for the heatable mounting frame)</td><td>Zeiss</td><td>411860-9010-000</td><td></td></tr><tr><td>Electroporation device</td><td>Amaxa</td><td>Nucleofector I</td><td></td></tr><tr><td>Fluorescence activated cell sorter</td><td>Becton Dickinson</td><td>FACSVantage SE</td><td></td></tr><tr><td>Image analysis software</td><td>Bitplane</td><td>7.0.0</td><td></td></tr></tbody></table>

real-time live imaging of t-cell signaling complex formation

防止传染病介导的免疫系统1,2。 T淋巴细胞的免疫系统的主协调，调节多个免疫细胞3,4的活化和反应。 T细胞活化是依赖于特定的抗原的识别由抗原呈递细胞（APC）显示。具体到每个T细胞克隆的T细胞抗原受体（TCR）和确定抗原特异性5。对抗原的T细胞受体的结合诱导的磷酸化的T细胞受体复合物的组成部分。为了促进T细胞活化，该信号必须转膜细胞质和细胞核，发起各种关键的反应，如招募到TCR信号蛋白APC网站（免疫突触），其分子活化，细胞骨架重排，细胞内钙离子浓度升高，并在6,7基因表达的变化。正确itiation和终止激活信号是至关重要的适当的T细胞应答。信号蛋白的活性依赖的形成和终止的蛋白质-蛋白质相互作用，翻译后的修改，如蛋白质磷酸化，形成的蛋白质复合物，蛋白泛素化和招聘的蛋白质，各种蜂窝基站的8。了解T细胞活化过程的内部运作是至关重要的免疫学研究和临床应用。 已经开发了各种测定为了研究蛋白质 - 蛋白质相互作用，然而，生化分析，如广泛使用的免疫共沉淀法，不使蛋白的定位待观察，从而排除了宝贵的见解的观察到的动态蜂窝机制。此外，这些批量的检测通常结合蛋白吨的不同阶段，从许多不同的细胞，可能会对他研究细胞过程。这可以有一个不利的影响时间分辨率。活细胞实时成像的使用允许空间跟踪的蛋白质和信号事件之间的时间分辨能力，从而脱落的过程9,10的动态光。我们提出一个方法的实时成像的T细胞活化过程中的信号复合物的形成。的主要的T细胞或T细胞系，如Jurkat细胞，被转染的质粒编码的单体荧光蛋白融合蛋白质的利益，防止非生理齐聚11。现场T细胞下跌超过盖玻片预先涂有T细胞活化抗体8,9，结合复杂的CD3/TCR的同时克服了需要为特定的活化抗原，诱导T细胞活化。活化细胞都在不断使用共焦显微镜成像。成像数据进行分析，以得到定量的结果，如山坳的信号蛋白的ocalization系数。

我们提出现场T细胞成像和分析的一个例子。成像实验之前，SLP76缺陷的T细胞（J14）确定的荧光蛋白的表达（ 图1）与使用的FACS分析。标签信令的蛋白质MCFP-NCK和SLP76 mYFP的的转染T细胞激活幻灯片和沉积在成像过程中T细胞的扩散（ 图2）。收集到的图像显示在整个激活和扩散（ 图3）的蛋白质的定位的动态。电脑图像处理提供了新的见解和定量信息的可视化细胞...

Watch this Scientific Journal Video about Real-time Live Imaging of T-cell Signaling Complex Formation at JoVE.com

T 细胞信号复合物形成的实时实时成像 |免疫学和感染|视频

Real-time Live Imaging of T-cell Signaling Complex Formation

我们描述一个活细胞成像的方法在T细胞活化过程中，提供了洞察蛋白质动力学。我们展示了结合使用的T细胞扩散法，激光共聚焦显微镜成像分析得到的定量结果，按照整个T细胞活化信号复合物的形成。

T细胞实时成像实时信号复合物的形成

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

real-time-live-imaging-of-t-cell-signaling-complex-formation

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13.6K 次观看.  Bar-Ilan University. 我们描述一个活细胞成像的方法在T细胞活化过程中，提供了洞察蛋白质动力学。我们展示了结合使用的T细胞扩散法，激光共聚焦显微镜成像分析得到的定量结果，按照整个T细胞活化信号复合物的形成。

视频: Real-time Live Imaging of T-cell Signaling Complex Formation

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

一个实时，同步成像的TCR和其相关的信号转导蛋白的T​​IRF显微镜技术

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

科研

免疫与感染

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the structure-function relationship of these receptors in situ, we need to visualize and track them on the live cell surface with enough spatiotemporal resolution. Here we show how to use recently developed Lattice Light-Sheet Microscopy (LLSM) to image T-cell receptors (TCRs) four-dimensionally (4D, space and time) at the live cell membrane. T cells are one of the main effector cells of the adaptive immune system, and here we used T cells as an example to show that the signaling and function of these cells are driven by the dynamics and interactions of the TCRs. LLSM allows for 4D imaging with unprecedented spatiotemporal resolution. This microscopy technique therefore can be generally applied to a wide array of surface or intracellular molecules of different cells in biology.

The goal of this protocol is to show how to use Lattice Light-Sheet Microscopy to four-dimensionally visualize surface receptor dynamics in live cells. Here T cell receptors on CD4+ primary T cells are shown.

使用莱迪思光片显微镜可视化表面 T-Cell 受体动力学四维

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

生物化学

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological synapses&#39;. Within these intimate cell-cell interfaces discrete sub-cellular clusters of MHC/Ag-TCR, F-actin, adhesion and signaling molecules form and remodel rapidly. These dynamics are thought to be critical determinants of both the efficiency and quality of the immune responses that develop and therefore of protective versus pathologic immunity. Current understanding of immunological synapses with physiologic APCs is limited by the inadequacy of the obtainable imaging resolution. Though artificial substrate models (e.g., planar lipid bilayers) offer excellent resolution and have been extremely valuable tools, they are inherently non-physiologic and oversimplified. Vascular and lymphatic endothelial cells have emerged as an important peripheral tissue (or stromal) compartment of &#39;semi-professional APCs&#39;. These APCs (which express most of the molecular machinery of professional APCs) have the unique feature of forming virtually planar cell surface and are readily transfectable (e.g., with fluorescent protein reporters). Herein a basic approach to implement endothelial cells as a novel and physiologic &#39;planar cellular APC model&#39; for improved imaging and interrogation of fundamental antigenic signaling processes will be described.

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 ...

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 ...

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

皮层肌动蛋白流通过结构照明显微镜数据的时空图像相关光谱量化性T细胞

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological synapse, a stereotyped cell-cell junction that regulates T cell activation and directionally targets effector responses. To study these processes effectively, an imaging approach that is tailored to capturing fast, polarized responses is necessary. This protocol describes such a system, which is based on a photoactivatable peptide-major histocompatibility complex (pMHC) that is non-stimulatory until it is exposed to ultraviolet light. Targeted decaging of this reagent during videomicroscopy experiments enables precise spatiotemporal control of TCR activation and high-resolution monitoring of subsequent cellular responses by total internal reflection (TIRF) imaging. This approach is also compatible with genetic and pharmacological perturbation strategies. This allows for the assembly of well-defined molecular pathways that link TCR signaling to the formation of the polarized cytoskeletal structures that underlie the immunological synapse.

This protocol describes an imaging-based method to activate T lymphocytes using photoactivatable peptide-MHC, enabling precise spatiotemporal control of T cell activation.

Photoactivatable 激动剂对 T 细胞活化的时空控制

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological ...

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 ...

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) ...

<meta charset="utf8"></head><body>用于检测 T 细胞中 Ca2+ 微结构域的高分辨率成像技术

Imaging Initial Ca2+ Microdomains in Primary T Cells

Local, sub-second Ca2+ signals, termed Ca2+ microdomains, are highly dynamic and short-lived Ca2+ signals, which result in a global [Ca2+]i elevation and might already determine the fate of a T cell. Upon T cell receptor activation, NAADP is formed rapidly, binding to NAADP binding proteins (HN1L/JPT2, LSM12) and their respective receptors (RyR1, TPC2) sitting on intracellular Ca2+ stores, like the ER and lysosomes, and leading to subsequent release and elevation of [Ca2+]i. To capture these fast and dynamically occurring Ca2+ signals, we developed a high-resolution imaging technique using a combination of two Ca2+ indicators, Fluo-4 AM and FuraRed AM. For postprocessing, an open-source, semi-automated Ca2+ microdomain detection approach was developed based on the programming language Python. Using this workflow, we are able to reliably detect Ca2+ microdomains on a subcellular level in primary murine and human T cells in high temporal and spatial resolution fluorescence videos. This method can also be applied to other cell types, like NK cells and murine neuronal cell lines.

<meta charset="utf8"></head><body>作者聚焦:揭示治疗干预的适应性免疫反应的初始激活

Here, we provide a comprehensive protocol to resolve initial local Ca2+ signals, known as Ca2+ microdomains, in primary murine and human T cells using fluorescence microscopy. This protocol serves as a valuable resource for researchers examining Ca2+ signaling pathways within immune cells and to further unravel their function.

原代 T 细胞中初始 Ca2+ 微结构域的成像

Local, sub-second Ca2+ signals, termed Ca2+ microdomains, are highly dynamic and short-lived Ca2+ signals, which result in a global [Ca2+]i elevation and ...

T细胞实时成像实时信号复合物的形成

摘要

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此视频中的章节

一个实时，同步成像的TCR和其相关的信号转导蛋白的TIRF显微镜技术

使用莱迪思光片显微镜可视化表面 T-Cell 受体动力学四维

成像免疫突触动力学内皮平面细胞模型

成像的CD4 T细胞间质迁移发炎真皮

皮层肌动蛋白流通过结构照明显微镜数据的时空图像相关光谱量化性T细胞

Photoactivatable 激动剂对 T 细胞活化的时空控制

人外周血和淋巴组织中原发性 T 细胞突触界面的评价

成像人类免疫学突触

原代 T 细胞中初始 Ca²⁺ 微结构域的成像

原代 T 细胞中初始 Ca²⁺ 微结构域的成像

T细胞实时成像实时信号复合物的形成

摘要

探索更多视频

此视频中的章节

一个实时，同步成像的TCR和其相关的信号转导蛋白的T​​IRF显微镜技术

使用莱迪思光片显微镜可视化表面 T-Cell 受体动力学四维

成像免疫突触动力学内皮平面细胞模型

成像的CD4 T细胞间质迁移发炎真皮

皮层肌动蛋白流通过结构照明显微镜数据的时空图像相关光谱量化性T细胞

Photoactivatable 激动剂对 T 细胞活化的时空控制

人外周血和淋巴组织中原发性 T 细胞突触界面的评价

成像人类免疫学突触

原代 T 细胞中初始 Ca2+ 微结构域的成像

原代 T 细胞中初始 Ca2+ 微结构域的成像

一个实时，同步成像的TCR和其相关的信号转导蛋白的TIRF显微镜技术

原代 T 细胞中初始 Ca²⁺ 微结构域的成像

原代 T 细胞中初始 Ca²⁺ 微结构域的成像