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>

<ol>
	<li>Viret, C., Janeway, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MHC+and+T+cell+development.">MHC and T cell development.</a> Rev. Immunogenet. 1, 91-104 (1999).</li><li>Risso, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+antimicrobial+peptides:+multifunctional+effector+molecules+of+innate+immunity.">Leukocyte antimicrobial peptides: multifunctional effector molecules of innate immunity.</a> J. Leukoc. Biol. 68, 785-792 (2000).</li><li>Doherty, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+T+cell+effector+and+memory+function+in+viral+immunity.">Cytotoxic T cell effector and memory function in viral immunity.</a> Curr. Top Microbiol. Immunol. 206, 1-14 (1996).</li><li>Jager, D., Jager, E., Knuth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+responses+to+tumour+antigens:+implications+for+antigen+specific+immunotherapy+of+cancer.">Immune responses to tumour antigens: implications for antigen specific immunotherapy of cancer.</a> J. Clin. Pathol. 54, 669-674 (2001).</li><li>Davis, M. M., Bjorkman, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell+antigen+receptor+genes+and+T-cell+recognition.">T-cell antigen receptor genes and T-cell recognition.</a> Nature. 334, 395-402 (1988).</li><li>Burkhardt, J. K., Carrizosa, E., Shaffer, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+actin+cytoskeleton+in+T+cell+activation.">The actin cytoskeleton in T cell activation.</a> Annu. Rev. Immunol. 26, 233-259 (2008).</li><li>Reicher, B., Barda-Saad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+pathways+leading+from+the+T-cell+antigen+receptor+to+the+actin+cytoskeleton+network.">Multiple pathways leading from the T-cell antigen receptor to the actin cytoskeleton network.</a> FEBS Lett. 584, 4858-4864 (2010).</li><li>Barda-Saad, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+molecular+interactions+linking+the+T+cell+antigen+receptor+to+the+actin+cytoskeleton.">Dynamic molecular interactions linking the T cell antigen receptor to the actin cytoskeleton.</a> Nat. Immunol. 6, 80-89 (2005).</li><li>Bunnell, S. C., Kapoor, V., Trible, R. P., Zhang, W., Samelson, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+actin+polymerization+drives+T+cell+receptor-induced+spreading:+a+role+for+the+signal+transduction+adaptor+LAT.">Dynamic actin polymerization drives T cell receptor-induced spreading: a role for the signal transduction adaptor LAT.</a> Immunity. 14, 315-329 (2001).</li><li>Balagopalan, L., Sherman, E., Barr, V. A., Samelson, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+techniques+for+assaying+lymphocyte+activation+in+action.">Imaging techniques for assaying lymphocyte activation in action.</a> Nat. Rev. Immunol. 11, 21-33 (2011).</li><li>Zacharias, D. A., Violin, J. D., Newton, A. C., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partitioning+of+lipid-modified+monomeric+GFPs+into+membrane+microdomains+of+live+cells.">Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells.</a> Science. 296, 913-916 (2002).</li><li>Adler, J., Parmryd, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+colocalization+by+correlation:+the+Pearson+correlation+coefficient+is+superior+to+the+Mander's+overlap+coefficient.">Quantifying colocalization by correlation: the Pearson correlation coefficient is superior to the Mander's overlap coefficient.</a> Cytometry A. 77, 733-742 (2010).</li><li>Costes, S. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+and+quantitative+measurement+of+protein-protein+colocalization+in+live+cells.">Automatic and quantitative measurement of protein-protein colocalization in live cells.</a> Biophys. J. 86, 3993-4003 (2004).</li><li>Fang, N., Motto, D. G., Ross, S. E., Koretzky, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tyrosines+113,+128,+and+145+of+SLP-76+are+required+for+optimal+augmentation+of+NFAT+promoter+activity.">Tyrosines 113, 128, and 145 of SLP-76 are required for optimal augmentation of NFAT promoter activity.</a> J. Immunol. 157, 3769-3773 (1996).</li><li>Wunderlich, L., Farag&#243;, A., Downward, J., Buday, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+Nck+with+tyrosine-phosphorylated+SLP-76+in+activated+T+lymphocytes.">Association of Nck with tyrosine-phosphorylated SLP-76 in activated T lymphocytes.</a> Eur. J. Immunol. 29, 1068-1075 (1999).</li><li>Pauker, M. H., Reicher, B., Fried, S., Perl, O., Barda-Saad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+cooperation+between+the+proteins+Nck+and+ADAP+is+fundamental+for+actin+reorganization.">Functional cooperation between the proteins Nck and ADAP is fundamental for actin reorganization.</a> Mol. Cell Biol. 31, 2653-2666 (2011).</li><li>Barda-Saad, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperative+interactions+at+the+SLP-76+complex+are+critical+for+actin+polymerization.">Cooperative interactions at the SLP-76 complex are critical for actin polymerization.</a> EMBO J. 29, 2315-2328 (2010).</li><li>Pauker, M. H., Hassan, N., Noy, E., Reicher, B., Barda-Saad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Studying the Dynamics of SLP-76, Nck, and Vav1 Multimolecular Complex Formation in Live Human Cells with Triple-Color. 5, rs3 (2012).</li></ol>

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?...

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			<td>Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Amaxa human T Cell nucleofector kit</td>
			<td>Lonza</td>
			<td>VCA-1002</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti CD3 (UCHT1 clone)</td>
			<td>BioLegend</td>
			<td>300432</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Falcon FACS Tubes</td>
			<td>Becton Dickinson</td>
			<td>352058</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>HyClone</td>
			<td>SV30160.03</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>G418</td>
			<td>Calbiochem</td>
			<td>345810</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>German coverglass system 4 chamber slides</td>
			<td>Lab-Tek II</td>
			<td>155382</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Bio Lab</td>
			<td>8410501</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Biological Industries</td>
			<td>03-025-1B</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hygromycin</td>
			<td>Enzo</td>
			<td>ALX-380-306</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Sigma</td>
			<td>G7513</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PBS (10X)</td>
			<td>Sigma</td>
			<td>D1408</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma</td>
			<td>P0781</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Poly-L-lysine 0.1% (w/v) in H2O</td>
			<td>Sigma</td>
			<td>P8920</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Sigma</td>
			<td>R8758</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma</td>
			<td>S2002</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Accublock digital dry bath</td>
			<td>Labnet</td>
			<td>D1105A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>Centrifuge 5810 R</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM 510 Meta</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Heatable mounting frame - Heating Insert P S</td>
			<td>PeCon</td>
			<td>130-800-031</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TempModule S (required for the heatable mounting frame)</td>
			<td>Zeiss</td>
			<td>411860-9010-000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electroporation device</td>
			<td>Amaxa</td>
			<td>Nucleofector I</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fluorescence activated cell sorter</td>
			<td>Becton Dickinson</td>
			<td>FACSVantage SE</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Image analysis software</td>
			<td>Bitplane</td>
			<td>7.0.0</td>
			<td>&nbsp;</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

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of &beta;-arrestins3. &beta;-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization 4-6.
Leukotriene B4 (LTB4) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB4 receptor 1 (BLT1; a high affinity receptor) and LTB4 receptor 2 (BLT2; a low affinity receptor)7-9. The LTB4-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis10-17. The current paper describes the methodologies developed to monitor LTB4-induced leukocyte migration and the interactions of BLT1 with &beta;-arrestin and , receptor translocation in live cells using microscopy imaging techniques18-19.
Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described 20-21. These cells were tested in live cell imaging methods to demonstrate LTB4 induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and &beta;-arrestin1 tagged with green fluorescent protein (&beta;-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &amp;#946;-arrestin

This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.

白三烯B实时成像 4介导的细胞迁移和BLT1与β- arrestin的相互作用

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

科研

免疫与感染

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 ...

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

在活体小鼠的血管炎症和血栓形成的异型血小板中性粒细胞被激活的内皮细胞相互作用对实时成像

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

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 ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

在髓系细胞动态实时成像<em&gt;装甲运兵车<sup&gt;最小/ +</sup</em&gt;肠道肿瘤通过旋转盘共聚焦显微镜

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Simultaneous Quantification of T-Cell Receptor Excision Circles (TRECs) and K-Deleting Recombination Excision Circles (KRECs) by Real-time PCR

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination process that creates T- and B-cell receptors. Because TRECs and KRECs are unable to replicate, they are diluted after each cell division, and therefore persist in the cell. Their quantity in peripheral blood can be considered as an estimation of thymic and bone marrow output. By combining well established and commonly used TREC assay with a modified version of KREC assay, we have developed a duplex quantitative real-time PCR that allows quantification of both newly-produced T and B lymphocytes in a single assay. The number of TRECs and KRECs are obtained using a standard curve prepared by serially diluting TREC and KREC signal joints cloned in a bacterial plasmid, together with a fragment of T-cell receptor alpha constant gene that serves as reference gene. Results are reported as number of TRECs and KRECs/106 cells or per ml of blood. The quantification of these DNA fragments have been proven useful for monitoring immune reconstitution following bone marrow transplantation in both children and adults, for improved characterization of immune deficiencies, or for better understanding of certain immunomodulating drug activity.

Simultaneous Quantification of T-Cell Receptor Excision Circles TRECs and K-Deleting Recombination Excision Circles KRECs by Real-time PCR

Here, we describe a method for simultaneous quantification of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs). The TREC/KREC assay can be used as marker of thymic and bone marrow output.

T细胞受体删除环同时定量（TRECs）和K-删除重组删除环（KRECs）通过实时PCR

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

在实时麻醉小鼠成像中性粒细胞和单核细胞在肠系膜静脉用活体显微镜

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

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 ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

使用成像流式细胞仪T细胞同种异体反应性测定

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

用两相循环流系统实时成像和定量真菌生物膜的研究

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...