这项工作已经得到了德意志研究联合会（DFG）BL567/3-1和弗里德里希·鲍尔基金会的资助。我们要感谢钱永健，在圣地亚哥加州大学的霍华德·休斯医学研究所的实验室为我们提供了标签RFP-T2。值得庆幸的是，我们承认，帕萨迪纳加州理工学院的戴维·巴尔的摩，和为我们提供的慢病毒质粒FUGW，psPAX2/pMD2.G，日内瓦，日内瓦大学Didier TRONO的。

该协议介绍TED应用细胞株，海马神经元及大脑皮质神经胶质细胞。 TED性能最好的时候，TED向量稳定表达，例如慢病毒载体。 TED的方法在图4中所示的示意性概观。 1。溶液的制备开始之前，应准备以下的解决方案，并可以被存储为指示。我们通常使用的玻璃和塑料材料和无菌水消毒。 <ol><li>准备HEPES林格（毫米）：125氯化钠，氯化钾，25 HEPES，2硫酸镁4 .7 H 2 O，2 氯化钙 ，1.25的NaH 2 PO 4·H 2 O，10 glucose.H 2 O无钙HEPES-林格（毫米）：25 HEPES，127氯化钠，氯化钾，硫酸镁4 .7 H 2 O 2，1.25的NaH 2 PO 4·H 2 O，10 glucose.H 2 O和0.1 EGTA。如果没有葡萄糖，这些成像解决方案可ê保存在4°C。新鲜使用前加入所需量的D-葡萄糖。 </li><li> TED分析海马神经元中，人工脑脊液（学联）建议。学联组成（毫米）：127 23氯化钠， 碳酸氢钠 ，氯化钾，3 2.5 NaHPO 4·H 2 O，25，D-glucose.H 2 O，2氯化钙，氯化镁2 .7 H 2 0在DDH 2 0。 </li><li>准备Fluo5N-AM至浓度为5mM。为了帮助增溶冻干Fluo5N-AM的50微克加入8.9微升20％的Pluronic F-127（在DMSO中，在室温下保存，防潮和避光保护）。然后，增溶Fluo5N-AM的至少2分钟的水浴超声波仪通过。店等分à在-20°C的0.5微升，从光照和水分保护。 </li><li>准备拮抗剂和激动剂股的需要。例如：）10毫米ATP或ADP（H 2 O，代谢激活嘌呤受体），B）10毫米卡巴在H 2 O（激动剂毒蕈碱型乙酰胆碱受体），C）30毫米环匹阿尼酸（CPA）在DMSO（SERCA阻滞剂），D）50MM DHPG PBS（代谢型谷氨酸受体激动剂代谢型谷氨酸受体I和代谢型谷氨酸受体5），E）10毫米谷氨酸在H 2 O，F）1毫米离子霉素在DMSO（载体），G）5毫米毒胡萝卜素DMSO（SERCA）高亲和力受体阻滞剂。在-20°C店等分</li><li>慢病毒载体：该协议包括TED慢病毒表达载体颗粒的使用。他们的生产和存储，是不是这个协议的一部分。我们使用重组羧酸酯酶的细胞系，神经胶质细胞和神经元的传输的第二代的慢病毒载体。我们的慢病毒系统基地37，表达载体FUGW和的的包装质粒pCMVΔR8.91或psPAX2的和假型质粒pMD2.G 38,39的 。 注意：考虑重组自失活的慢病毒载体中的工作要求仔细考虑生物安全指引。在许多国家，这些向量被归类为第2组的生物危害风险。欲了解更多信息，请参阅<a href="http://www.addgene.org/lentiviral" target="_blank">http://www.addgene.org/lentiviral/</a> 。 </li></ol> 2。制备与病毒感染的小鼠海马神经元<ol><li>在玻璃培养皿中的直径为20厘米（每皿100盖玻片）1个约30分钟70％的乙醇洗涤盖玻片。最后，加100％的乙醇（PA）2分钟。去除残留的乙醇和无菌罩下干燥的盖玻片。 </li><li>准备海马神经元培养两种不同类型的媒体：（一）Neurobasal培养基，B27 1:50（二）全介质：Neurobasal培养基，B27 1:50，1:100 GLUTAMAX，和N2补充1:100。无菌滤液的介质，并将其储存在细胞培养箱中，直到使用。 </li><li>清扫前一天准备首先在各放置一个无菌的10毫米盖玻片，盖玻片以及4孔细胞培养皿。之后，仔细大衣每个盖玻片80-100微升0.1％聚-L-赖氨酸和存储的菜培养箱中过夜。 </li><li>在解剖和细胞培养的日子，开始与100微升的HBSS洗涤盖玻片三次到每个盖玻片和转让100微升的neurobasal介质。将菜平衡的孵化器（ 例如在解剖小鼠）。 </li></ol>解剖我们执行我们的实验小鼠按照欧盟指引，我们的机构的动物护理和利用委员会批准。 <ol start="5"><li>卸下总大脑和解剖海马双边。 </li><li>小心取出任何脑膜及其他组织超过海马和地方一海马各1.5毫升管含有450微升HBSS。存储足够数量的海马组织置于冰上，直到已被隔离。 </li></ol> &lt;P类=的“jove_step”&gt;细胞培养<ol start="7"><li>每管加入50微升1％胰蛋白酶（沃辛顿），孵育在37℃水浴15分钟。偶尔摇动试管。要停止消化反应，每管加入50微升1％的胰蛋白酶抑制剂和反转管数次。 </li><li>所有组织转移可达5海马一个15毫升隼避免了输送液体的管。加入B27-介质（a），至终体积为5ml。 </li><li>磨碎组织使用火抛光的巴斯德玻璃吸管小心吹打向上和向下。 注意：避免气泡！ </li><li>自旋用400×g离心3分钟，吸出上清液，并在5ml B27-介质（a）中重悬细胞沉淀。 </li><li>再次用玻璃吸管组织磨碎，然后两次与1000微升塑料过滤嘴的帮助下。执行中所描述的步骤2.9和2.10。 </li><li>后的最后离心，重悬细胞沉淀在毫升全mediu 2“米（b）和磨碎细胞与200μl的塑料过滤嘴。从单细胞悬液分离剩余的细胞团块，让细胞团块安顿下来1-2分钟。 </li><li>的细胞进行计数，并计算所需要的病毒感染的细胞的总数。对于TED成像，使用25,000个细胞，每10毫米盖玻片。 </li><li>电镀额外25,000非转导细胞作为对照，建议在这一点上。 </li></ol>病毒感染<ol start="15"><li>病毒感染的细胞悬液转移到一个新的15毫升隼管，在400×g离心3分钟。 </li><li>吸出上清液，并在300微升培养基悬浮细胞（B） </li><li>添加适量的传染性TED慢病毒表达载体颗粒，并，让猎鹰管在室温下放置10分钟。 </li><li>填补了全介质（B）最终体积需要电镀（100微升，每10毫米盖玻片）管，吸介质覆盖erslip，立即将100微升细胞悬液到每个盖玻片。 </li><li>进入孵化器将菜，直到所有的细胞已经安顿下来（约2小时），并认真填写了碗筷，直到它们含有2毫升培养基（B）。 </li><li>让神经细胞生长至少一个星期。更换50％的培养基中每星期。 </li></ol> 3。文化和病毒感染的小鼠皮层神经胶质细胞准备<ol><li>首先通过制备基底神经胶质细胞培养基（DMEM/F12 1:1混合物中有10％FCS，5％HS，1％青霉素/链霉素，0.45％葡萄糖）和涂层的T75细胞的培养瓶中，用4ml 0.5 ng / ml的聚DL-鸟氨酸氢溴酸（色情）（稀释后在150毫米的硼酸溶液，pH值8.35）。后，在37℃下温育2小时或过夜，洗涤细胞培养瓶中用HBSS三次，并加入18 ml的的基底神经胶质细胞培养基中含有B27 1:50，10 ng / ml的EGF。平衡在孵化器中（最多3小时）的细胞培养瓶中。 </li></ol> &lt;P类，=“jove_step”&gt;解剖<ol><li>我们执行我们的实验小鼠按照欧盟指引，我们的机构的动物护理和利用委员会批准。 </li><li>解剖一个P5-P7小鼠的大脑取出海马和脑膜从两个半球2.5。 </li><li>解剖额叶皮层，它切成若干小块，并收集他们在1.5毫升含有HBSS管。管存放在冰上进行细胞培养部分。 </li></ol>细胞培养<ol start="4"><li>从管所有组织转移到15毫升猎鹰管使用火抛光的玻璃吸管。 </li><li>基础培养基中添加5毫升至终体积和磨碎组织吹打几次火抛光的玻璃吸管向上和向下。在300×g离心3分钟后，离心，吸出培养基，并在5ml基本培养基中重悬细胞沉淀。 </li><li>重复步骤3.5的两倍。 </li><li>后氏三次离心，重悬细胞沉淀在2ml的基底神经胶质细胞培养基中含有B27（1:50）和10 ng / ml的EGF。 </li><li> Titruate再次转移的细胞悬浮液制备的T75细胞的培养瓶中，让细胞生长3-4天。 </li></ol>洗胶质细胞（3-4天中文化后）： <ol start="9"><li>用10ml PBS洗涤细胞一次，并大力挖掘或轻轻打烧瓶用你的手，一边拿着紧张，另一方面几次。通过该工序的细胞碎片和细胞团从培养物中除去。 </li><li>吸PBS和添加新鲜的基底神经胶质细胞的培养基中含有B27（1:50）和10 ng / ml的EGF。进一步培育文化为5-7天，直到细胞达到80％汇合。 </li></ol>拆分，转导和最终播种的神经胶质细胞： <ol start="11"><li>外套10毫米盖玻片用100微升聚-D-赖氨酸（联合解决方案：0.1％，稀释1/50广告PBS或HBSS 20微克/毫升），并培育培养箱中过夜。清洗盖玻片HBSS三次加100微升基底神经胶质细胞介质。在孵化器平衡盖玻片（3小时）。 </li><li>两次用10ml PBS洗胶质细胞，吸出PBS，并加入3毫升胰蛋白酶（TrypLE，未稀释）。胰蛋白酶消化细胞，最长为1分钟。 注意：避免过度胰蛋白酶。一般情况下80％以上的所有单元格的1分钟后取下，并有几百万的健康细胞，这是足够的。 </li><li>停止反应，加入10ml基底神经胶质细胞介质，以300×g将细胞悬浮液转移到15毫升隼管，离心3分钟。吸出上清液，重悬细胞沉淀在5毫升的的基底神经胶质细胞介质。 </li><li>同样地，旋转和吸出步骤3.15中所描述的，然后细胞悬浮在5ml的基底神经胶质介质。 </li><li>所需数量的细胞转移到1.5 ml管。 10 4 2×10 4胶质细胞是足够一个10毫米盖玻片。一般，将悬浮液体积UME转导细胞4菜是小于200微升。添加适量的慢病毒式表达载体颗粒的细胞，并在RT下孵育10分钟。然后填写暂停基底神经胶质细胞中播种量每盖玻片）（100微升。 </li><li>种子100微升每盖玻片和大约2小时的孵育感染的细胞，再加入的基底神经胶质细胞培养基中含有B27 1:50，10 ng / ml的EGF，至终体积为2ml。 </li><li>对于理想的培养条件下使用代细胞用于实验电镀后3-7天。 </li></ol> 4。记者株的产生与文化 TED记者细胞系建立的基础上，BHK21的HeLa，HEK293和SH-SY5Y细胞。这里，我们提供的HeLa细胞的协议，但该协议可能被转移到任何其他的细胞系。 生成稳定的HeLa细胞系<ol><li>拆分的HeLa细胞系野生型和转100,000ç在体积为200微升的1.5毫升管中的细胞l。 </li><li>添加适量的慢病毒式表达载体颗粒的试管中，并在RT下的10分钟孵育细胞病毒悬浮液。然后种子细胞的总体积为2毫升培养基（DMEM，10％FCS，1％青霉素/链霉素）中为30毫米的菜，孵育3天。 </li><li>用PBS洗涤一次后，吸介质和添加500μL胰蛋白酶（TrypLE，在PBS 2:3）。孵育约。 3分钟，然后加入3毫升培养基停止反应。转移到15毫升猎鹰管和离心3分钟，在400 XG暂停。 </li><li>在200μl培养基中重悬细胞沉淀，并再次用慢病毒颗粒感染细胞，如4.2中所述。然后，在培养瓶中，在10毫升培养基的T25细胞的种子细胞。 </li><li>细胞生长，直到达到60-80％汇合。然后分裂的文化。 </li></ol> TED记者细胞株<ol start="6"><li> TED记者的细胞系可以维持在使用任何标准的介质中， 例如含5％或10％FCS和1％青霉素/链霉素。 </li><li>铠适当数量的细胞在含4孔盘的无菌的10毫米的盖玻片（见上文）， 例如，10,000至20,000个细胞每盖玻片。 </li><li>细胞生长至少两天才使用他们的TED成像。 </li></ol> 5。在倒置显微镜的实时成像过程的准备<ol><li><ol class="lalpha"><li> prewarm HEPES林格氏液至室温，并加入D-葡萄糖。 </li><li> prewarm ACSF（不含Ca 2 +和Mg 2 +）至室温，并加入D-葡萄糖。 5分钟通气与CARBOGEN学联。添加1M的CaCl 2和MgCl 2的终浓度为2mM的储备液。 </li></ol></li><li>启动实时成像显微镜和所有的计算机程序。 </li><li>开始灌注一个“虚拟”成像室，平衡管系统。</li><li> Prewarm内嵌在显微镜载物台的解决方案的加热器和成像室。修复成像室，在加热插入。 </li><li>平衡系统到32-37°C，然后再开始染料的加载过程。 注意：为了避免意外流灌注液使用显微镜系统，我们的目标和弹性硅胶片（1毫米），以保护周围头发的关系显微镜阶段。 </li></ol> 6。无论是细胞类型的染料装载<ol><li>重悬在100μl预热的成像解决方案（HEPES林格氏或学联）1等份的Fluo5N-AM（见1.3）。 </li><li>溶解Fluo5N-AM在成像液（HEPES-林格氏或ACSF）在水浴超声波仪，持续90秒。 </li><li>使用新鲜的4 - 或24孔板和转移400μl的预热的成像解决一个阱。添加染料溶液中，以同井500μl的5微米的解决方案来。 </li><li>小心地放置盖玻片细胞（ 例如直径10-18毫米）以及细胞朝上。 </li><li>孵育一段适当的时间在细胞培养孵化器在37°C（同时准备成像显微镜阶段设置）。典型的染料加载时间为神经元和神经胶质细胞7-15分钟，细胞株10-20分钟。 </li></ol> 关键：染料加载时间和染料浓度取决于细胞类型，细胞密度，实验的具体需求！很长的反应时间，在染料的存在下，可能会损害细胞，尤其是原代神经元原代神经胶质细胞和生理刺激的响应，这是至关重要的。长潜伏期时间（ 如 30分钟）只会是有益的ER钙本地化实验调查ER钙分布在小亚细胞结构， 如细 ​​小突起或棘。在一些实验中，混合介质与成像解决方案的1/3的增长，可能有助于保护细胞在Fluo5N-AM加载。 <ol start="6"><li>瞬间，盖玻片转移到另一个细胞培养以及含有成像解决方案（HEPES林格氏或学联），并开始安装细胞成像室。 </li></ol> 7。 ER-钙活细胞成像一个倒激光扫描共聚焦显微镜<ol><li>使用油漆刷成像室申请高真空润滑脂。的润滑脂，需要注视到灌注腔室的底部的盖玻片。我们使用自制成像室为10-12毫米盖玻片具有体积小，从而使高灌注速度可达每分钟15倍缓冲交换。 18毫米盖玻片，市售灌注室可购买从华纳工具（RC-49FS）。该室还使神经元的刺激。 </li><li>拿起盖玻片与载Fluo5N细胞和成像解决方案（50-100微升）加一小滴，到细胞，保持细胞成像解决方案覆盖。将盖玻片倒置，安装细胞成像室。按盖玻片到硅胶，使用棉签（ 如 Q-提示）。 </li><li>从底部的玻璃盖玻片，用棉签擦去残留的缓冲注意：，仔细擦去残留的水分，当使用油高数值孔径的物镜高分辨率成像。残留的盐是最好的水除去。意外涂抹油脂可能会被删除用丙酮或纯乙醇。 </li><li>交易所“虚拟”成像与实验成像室室。 5-10分钟的连续灌注清洗细胞。 </li><li>洗涤细胞5 - 10分钟连续灌注。 </li><li>用于小区选择使用最小量的激光照射，高扫描速率（2-4赫兹），一个小的图像尺寸，和高增益。 </li><li> 注意：对于选择的Fluo5N标记细胞，不使用光或直接照明epifluorescen的超过吨光。明亮的照明的Fluo5N/Ca 2 +复合物会导致完全丧失雌激素受体 ​​特异性荧光在2-4秒之内（ 图5）。 </li><li>显微镜，根据实验计划。方向，代表不同的式载体中的摄像设置表1中给出。对于倒激光扫描共聚焦显微镜，我们使用与Olympus IX81显微镜与Fluoview 1000配备二极管激光器（15毫瓦473纳米，559纳米，20毫瓦）和光谱的检测器系统的共聚焦系统相结合。 </li><li>在开始实验文件感兴趣的细胞通过高分辨X，YZ图像栈。 </li><li>在特定的实验条件下进行X，YT成像监视ER钙动力学。我们的系统的典型设置列于表2。 </li><li>监控改变ER钙下连续灌注成像解决方案。典型的缓冲区汇率是电子xemplarily：（一）SERCA块的的细胞清洗和存储消耗：1.5毫升/分钟;（二）ATP / DHPG刺激的：3毫升/分钟。 </li><li> 12位（4096灰度值）优先获取数字图像。对于并行成像的Fluo5N/Ca 2 + RFP，8位图像（256级灰度值）可能会拯救你的系统，从数据溢出。 </li><li> ImageJ的兼容格式（ 如 TIFF）存储在活细胞的时间序列图像（X，YT）或3D图像栈（X，YZ）（细胞分布Fluo5N/Ca 2 +复合物）。 </li></ol> 8。图像处理和数据分析<ol><li> ImageJ的程序中打开图像堆栈。在多色成像的情况下，分割的RGB通道当记者问ImageJ的。 </li><li>识别您的权益回报率（ROI）的区域（次）仔细检查图像栈（ 例如帮助的强度与时间曲线） </li><li>使用时间系列分析仪插件读出投资回报率（ 原料 ）的平均像素强度。 ，取决于克的目的，完整的ER ER 如 ER树突周边小区的区域或小区域周围绘制的ROI。还包括背景减法无细胞1-3领域的投资回报率。 </li><li>背景的投资回报率平均荧光值计算，从F 原始值减去背景值F B得到的F ROI值计算的平均背景荧光（F B）。 </li><li>计算出F 0，它是基底细胞的荧光，通过计算10到30处于休息状态的每个ROI的荧光值的平均值。 </li><li>的背景校正后的相对荧光变化ΔF/ F 0由下式计算： </li></ol><img alt="公式1" fo:content-width="1.5in" fo:src="/files/ftp_upload/50317/50317eq1highres.jpg" src="/files/ftp_upload/50317/50317eq1.jpg" /><ol start="7"><li>目前的相对变化荧光NCE作为跟踪与ΔF/ F为0的X轴的Y轴，以及时间。 </li></ol>

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J., Baltimore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+transmission+and+tissue-specific+expression+of+transgenes+delivered+by+lentiviral+vectors.">Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors.</a> Science. 295, 868-872 (2002).</li><li>Zufferey, R., 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=Self-inactivating+lentivirus+vector+for+safe+and+efficient+in+vivo+gene+delivery.">Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery.</a> Journal of Virology. 72, 9873-9880 (1998).</li><li>Zufferey, R., Nagy, D., Mandel, R. J., Naldini, L., Trono, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiply+attenuated+lentiviral+vector+achieves+efficient+gene+delivery+in+vivo.">Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo.</a> Nature Biotechnology. 15, 871-875 (1997).</li><li>Li, D., Herault, K., Oheim, M., Ropert, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FM+dyes+enter+via+a+store-operated+calcium+channel+and+modify+calcium+signaling+of+cultured+astrocytes.">FM dyes enter via a store-operated calcium channel and modify calcium signaling of cultured astrocytes.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 21960-21965 (2009).</li><li>Blum, R., Petersen, O. H., Verkhratsky, A., Verkhratsky, A., Petersen, O. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Calcium measurement methods. Vol. Neuromethods 43 (Springer protocols), 147-167 (2010).</li><li>Tian, L., 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=Selective+esterase-ester+pair+for+targeting+small+molecules+with+cellular+specificity.">Selective esterase-ester pair for targeting small molecules with cellular specificity.</a> Proceedings of the National Academy of Sciences of the United States of America. 109, 4756-4761 (2012).</li><li>Samtleben, S., Blum, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+vectors+for+direct+ER+calcium+imaging+with+targeted-estersase+induced+dye+loading.">Improved vectors for direct ER calcium imaging with targeted-estersase induced dye loading.</a> , (2012).</li></ol>

作者宣称，他们有没有竞争经济利益。

TED的方法的优点和缺点已进行了广泛的讨论，在最近的出版物34,35。 TED情况下，与上述的其他方法相比，细胞破坏和灌注的问题。此外，需要强调两个方面。 TED ER钙成像的原则要求（1）目标TED的载体构建的帮助下，内质网腔，和（2）的高效率和低亲和力的合成AM酯优先释放活性羧酸酯酶表达内质网腔的钙染料。 1。 TED载体构建 图3总?...

为了解决ER钙的生理反应，我们开发了一个新的策略，以提高合成钙敏感染料进入ER诱捕。该方法能够直接的，非破坏性的实时监控自由ER钙在细胞外钙离子的存在。 ER钙的功能和信令钙信号被发现的不同类型的细胞， 如肌肉细胞，神经元和神经胶质细胞，其功能范围从调解肌肉收缩，参与突触传递的学习和记忆1,2。游离钙离子浓度的变化有很高的科学价值，因为钙参与调节基因转录，细胞增殖，神经细胞的兴奋性，细胞死亡和其他细胞信号事件1-7。所有这些细胞钙信号连接功能，并有助于细胞内钙存储动态8-10。 所有的钙信号中的一个共同特点是钙外空间之间的流动，细胞质和细胞器，主要是ER和线粒体。这会导致这些细胞器内的钙离子浓度的动态变化，由不同的信令组件感测。一般情况下，内质网中的钙浓度的范围在100至800微米之间，在细胞质中的钙离子浓度接近100纳米，和在细胞外空间中的浓度大约是1-2毫米。因此，存在很高的化学驱动力细胞质中的钙离子流向2,9,10。 最常研究的ER-源性钙信号依赖于G-蛋白偶联受体（GPCR），然后激活磷脂酶C（PLC）的刺激。反过来，PLC在生产肌醇1,4,5 -三磷酸肌醇（IP 3）1。 IP 3结合后其REC在ER膜的eptor（IP 3， 图1（记录）），钙离子从内质网腔中释放。在历史上，第一IP-3介导从内质网释放钙-间接即使-测量胰腺腺泡细胞由Streb 等的。于1983年11。此出版物建议在第一次涉及乙酰胆碱，磷脂酶C和IP 3的信号级联。这种方式的钙离子的释放一般被称为IP 3引起的钙离子的释放（IiCR）（ 图1）。依赖激酶激活磷脂酶Cγ受体酪氨酸激酶生长因子和神经营养因子的作用ER钙信号后IP海拔3 12。钙海拔在除了IiCR，可介导的离子型钙条目，例如兰尼再通过电压门控钙通道（CA V），以及随后的钙诱导钙释放（CICR）与其受体（RyR的）。 IiCR和CICR生理链接库操纵的钙进入（SOCE）。 SOCE包括STIM（基质相互作用的分子），这是一个传感器，用于ER钙离子释放的动作。激励信号已被证明，通过瞬时受体电位通道色氨酸（Trp），13，14 Orai钙离子通道和电压门控钙离子通道15（ 图1）刺激细胞外钙离子条目。 ER钙丢失是动态救出由的肉瘤内质网钙ATP酶（SERCA），积极的泵钙背到急诊室的作用。阻断药物，如毒胡萝卜素SERCA ER钙胞质室推出了一个连续亏损。该ER钙“泄漏”Sec61蛋白复合物16,17（ 图1）配合物，如ER膜内孔隙所造成的。 贝里奇1998年，出版了一本模型中，“神经元内的神经元模型”，这ħ建议的原则生理作用的ER结合神经元钙5。该模型认为，存在一个连续的的ER膜系统形成一个细胞内的“形象”的神经细胞质膜5。这个二进制真核生物膜系统是自称是快与慢的神经元钙信号的时间与空间整合的基本前提。钙信号发生随之而来，随后在不同的相同的神经元树突棘或赋予细胞的胞体或细胞核通过急诊室，在那里他们总结了5,18。然后，其总和可能会影响神经元的兴奋性，调节基因转录或集成的信号级联。因此，ER支持钙信号的整合。这一概念的一个先决条件是所声称的一些研究，并已被证明至少为躯体-D在单个细胞中的雌激素受体的连续性endritic地区和短距离轴突预测19-21。是否有内长轴突预测ER连续性是一个有争议的问题。 来测量其流量超过ER膜游离钙的战略钙信号的最频繁监测在细胞质中22,23。因此，它可以很容易地区别是否的Ca 2 +流进入细胞质从细胞外或细胞内储存6,24。为了克服这个限制，方法策略直接ER钙成像已经制定出来。总之，使用下列策略：（1）ER-针对性的基因工程蛋白质指标25-27。基于蛋白质的低亲和力的Ca 2 +的指标，使用的生物发光蛋白水母发光蛋白结合钙传感蛋白或GFP。这些基因工程的Ca 2 +的指标（GECIS）定位到内质网的信号肽的帮助下，积极地保持在ER中使用的保留和检索动机。常见的ER的Ca 2 +指标上卡梅里安原则基础卡默莱昂YC4.3 26,28; 29分裂YC7.3ER卡梅里安，卡梅里安D1 30。 （2） 直接酯酶基染料装载AM酯低亲和力的Ca 2 + 指标 31,32 AM衍生工具的指示剂染料（MAG-Fura2-AM，MAG-Fluo4-AM-AM Fluo5N）通过生物膜的亲脂性，钙不敏感的状态。然后，在细胞质中，以及在ER中，内源性酯酶对AM-酯基裂开，释放的Ca 2 +指示剂，留下一定量的活性染料在细胞质中，在ER。因此，这种方法在ER中的钙离子浓度高的条件下是有用的，只要细胞内游离钙离子的浓度保持低于去低亲和性的指标的TECTION限制，特别是在特征钙信号（ 例如，纳米低μM）。 （3）AM酯组合质膜通透性32加载，任何剩余的胞浆Ca 2 +指示剂除去质膜通透性与少量的“温和”的清洁剂（ 例如皂甙）在人工细胞内的缓冲。因此，细胞内的膜的可能的刺激， 例如 ，与在细胞内的缓冲液中的IP 3，可直接通过质膜中的“孔”。 （4） 胞液中透析，根据全细胞配置和同时测量的Ca 2 +在ER腔体和的胞质溶胶32,33。细胞被第一次装载有低亲和力的Ca 2 +的指示器（ 例如，幅度- Fura2上午，比例，UV光）。之后，与帮助的补丁吸管，任何其它细胞色素osolic低亲和性的Ca 2 +指示剂透析的胞液中的缓冲液含有高亲和力的Ca 2 +的指示器（ 例如萤光-3，可见光）。这一战略使细胞质和ER派生的信号同步记录。 （5） 目标酯酶诱导染料加载 8,34。甲羧酸酯酶（CES）上被定位到内质网的内腔中，并提供了一个高的酯酶活性对AM-酯的形式存在的低亲和力的Ca 2 +的指标的有效捕获。 有针对性的酯酶诱导染料吸附量（TED） 为了提高针对低亲和力的Ca 2 +的指标内质网腔，TED的开发。 TED需要一个ER靶向小鼠羧酸酯酶（CES2）（ 图2），这是实现通过表达构建的过表达。对AM-酯的形式存在的钙孵育细胞表达重组CES构造的dicator染料（Fluo5N-AM， 图2）。然后，在ER中，染料被转换到的Ca 2 +敏感的，不透膜的Ca 2 +指示剂络合物（Fluo5N/Ca 2 +）的高的酯酶活性，因此，捕获的内质网腔8中以高浓度的染料，34。该方法是特别有用的调查通过IiCR途径例如雌激素受体 ​​的钙释放，通过代谢，嘌呤或谷氨酸受体8,34和可视化ER钙耗竭后，在SERCA 17的封锁，通过“泄漏通道”，例如直接，34。据我们的经验，低亲和力的Ca 2 +指标Fluo5N-AM是目前最好的可用指标，使用TED的染料加载策略。 Fluo5N-AM具有低亲和力的Ca 2 +（解离常数K D〜90微米， 图2），其AM-形式几乎无荧光，但提供高荧光发射后calciu米8,34约束力。 〜490 nm处，对应于标准染料如FITC标记的Alexa 488，或绿色荧光蛋白与一个标准的光源，可兴奋的Fluo5N/Ca 2 +配合物。细胞内钙信号几乎达到在低浓度μM范围内，因此，几乎没有检测到在细胞质中Fluo5N 35。 为了提高的TED性能，开发一些重组载体构建（ 图3）。本来，式载体的基础上的编码序列（RefSeq的登记号为NM_145603，CES2c CES2）和最佳的TED性能是观察到与CES的结构稳定表达。新TED载体表达红色荧光蛋白TagRFP-T2 36 CES2融合的核心要素。这些载体的优点是，它们可以被用来识别转导的细胞，使用的红色荧光变化的归一化在Fluo5N/Ca 2作为内部控制+荧光。红色荧光还提供了可能性，以可视化的刺激条件下的ER和ER动态变化的结构分布。

<table border="1">
	 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>(S)-3,5-DHPG (Dihydroxyphenylglycine)</td>
 <td>Tocris</td>
 <td>0805</td>
 <td></td>
 </tr>
 <tr>
 <td>5';-ATP-Na2, ATP disodium salt hydrate</td>
 <td>Sigma</td>
 <td>A26209-1G</td>
 <td></td>
 </tr>
 <tr>
 <td>B-27 supplement,50x</td>
 <td>Invitrogen</td>
 <td>17504-044</td>
 <td></td>
 </tr>
 <tr>
 <td>Carbamoylcholine chloride (Charbachol)</td>
 <td>Sigma</td>
 <td>C4382-1g</td>
 <td></td>
 </tr>
 <tr>
 <td>Cyclopiazonic acid (CPA)</td>
 <td>Ascent scientific</td>
 <td>Asc-300</td>
 <td></td>
 </tr>
 <tr>
 <td>Dimethylsulfoxide (DMSO)</td>
 <td>Sigma</td>
 <td>D2650</td>
 <td></td>
 </tr>
 <tr>
 <td>DMEM with Glutamax</td>
 <td>Invitrogen-Gibco</td>
 <td>31966-047</td>
 <td></td>
 </tr>
 <tr>
 <td>Dulbecco's PBS without Ca/Mg 1x</td>
 <td>PAA</td>
 <td>H15-002</td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal calf serum</td>
 <td>Invitrogen-Gibco</td>
 <td>10270-106</td>
 <td></td>
 </tr>
 <tr>
 <td>Fluo-5N-AM, 10 x 50 &mu;g</td>
 <td>Invitrogen</td>
 <td>F14204</td>
 <td></td>
 </tr>
 <tr>
 <td>Glutamate</td>
 <td>Ascent scientific</td>
 <td>Asc-049</td>
 <td></td>
 </tr>
 <tr>
 <td>Glutamax</td>
 <td>Invitrogen</td>
 <td>35050-038</td>
 <td></td>
 </tr>
 <tr>
 <td>Ham's F12 nutrients mixture</td>
 <td>Invitrogen-Gibco</td>
 <td>21765-029</td>
 <td></td>
 </tr>
 <tr>
 <td>Hank's BSS(1x) without Ca,without Mg, with Phenol Red, HBSS</td>
 <td>PAA Laboratories</td>
 <td>H15-010</td>
 <td></td>
 </tr>
 <tr>
 <td>Horse serum</td>
 <td>SHD3250YK</td>
 <td>Linearis</td>
 <td></td>
 </tr>
 <tr>
 <td>Ionomycin Ca2+ salt</td>
 <td>Ascent scientific</td>
 <td>Asc-116</td>
 <td></td>
 </tr>
 <tr>
 <td>murine EGF </td>
 <td>PreproTech</td>
 <td>315-09</td>
 <td></td>
 </tr>
 <tr>
 <td>N2 supplement 100x</td>
 <td>Invitrogen</td>
 <td>17502-048</td>
 <td></td>
 </tr>
 <tr>
 <td>Neurobasal medium</td>
 <td>Invitrogen-Gibco</td>
 <td>21103-049</td>
 <td></td>
 </tr>
 <tr>
 <td>Pluronic F127, low UV absorbance,2g</td>
 <td>Invitrogen</td>
 <td>P6867</td>
 <td></td>
 </tr>
 <tr>
 <td>Poly-DL-ornithine hydrobromide PORN</td>
 <td>Sigma</td>
 <td>P8638</td>
 <td></td>
 </tr>
 <tr>
 <td>Poly-D-lysine hydrobromide</td>
 <td>Sigma</td>
 <td>P7280</td>
 <td></td>
 </tr>
 <tr>
 <td>Poly-L-Lysin</td>
 <td>Sigma</td>
 <td>P2636</td>
 <td></td>
 </tr>
 <tr>
 <td>Thapsigargin</td>
 <td>Calbiochem</td>
 <td>586005-1mg</td>
 <td></td>
 </tr>
 <tr>
 <td>TryLE Express</td>
 <td>Invitrogen</td>
 <td>12605-028</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypsin</td>
 <td>Worthington</td>
 <td>LS-003707</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypsininhibitor from Glycine MAX (soybean)</td>
 <td>Sigma</td>
 <td>T6522</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Materials</td>
 </tr>
 <tr>
 <td>Confocal system: inverted laser scanning microscope: FV1000 with IX81</td>
 <td>Olympus</td>
 <td></td>
 <td>user-specific configuration</td>
 </tr>
 <tr>
 <td>Confocal system: laser combiner FV10 and diode lasers (405, 473, 559, 635)</td>
 <td>Olympus</td>
 <td></td>
 <td>user-specific configuration</td>
 </tr>
 <tr>
 <td>Confocal system: scan head FV10-SPD with spectraldetector</td>
 <td>Olympus</td>
 <td></td>
 <td>user-specific configuration</td>
 </tr>
 <tr>
 <td>Heater controller TC-344B</td>
 <td>Warner Instruments</td>
 <td>64-0101</td>
 <td>to control in line solution heater</td>
 </tr>
 <tr>
 <td>NIH ImageJ software</td>
 <td>WS Rasband, ImageJ, US National Institutes of Health, <a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a>, 1997--2006</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Objective: UAPON20XW340 NA 0,7 WD 0,35</td>
 <td>Olympus</td>
 <td>N2709100</td>
 <td></td>
 </tr>
 <tr>
 <td>Objective: UPLFLN40XO</td>
 <td>Olympus</td>
 <td>N1478700</td>
 <td></td>
 </tr>
 <tr>
 <td>Objective: UPLSAPO60XO/1.35, WD=0.15</td>
 <td>Olympus</td>
 <td>N1480700</td>
 <td></td>
 </tr>
 <tr>
 <td>Perfusion chamber, inverse</td>
 <td>custom-made</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Perfusion chamber, RC-49FS</td>
 <td>Warner Instruments</td>
 <td>W4 64-1709 </td>
 <td></td>
 </tr>
 <tr>
 <td>Perfusion tube: PT-49</td>
 <td>Warner Instruments</td>
 <td>64-1730</td>
 <td>for perfusion</td>
 </tr>
 <tr>
 <td>Peristaltic pump MiniPlus 3 (4 channels)</td>
 <td>Gilson</td>
 <td>n.a. </td>
 <td>perfusion pump</td>
 </tr>
 <tr>
 <td>Single inline solution heater SH-27B</td>
 <td>Warner Instruments</td>
 <td>64-0102</td>
 <td>controlled by heater controller</td>
 </tr>
 <tr>
 <td>Suction tubes: ST3R</td>
 <td>Warner Instruments</td>
 <td>64-1407</td>
 <td>for perfusion</td>
 </tr>
 <tr>
 <td>Heating insert: Tempocontrol 37-2 digital 2-channel</td>
 <td>Pecon</td>
 <td>n.a. </td>
 <td></td>
 </tr>
	 </tbody>
 </table>

direct imaging of er calcium with targeted-esterase induced dye loading (ted)

重要的是要了解钙的作用在细胞生理钙动态的可视化。要检查钙动力学，合成的Ca 2 +荧光指示：已成为流行。在这里，我们展示了TED（=有针对性的酯酶诱导染料吸附量），一种方法来提高释放的Ca 2 +指示剂染料在不同类型的细胞内质网腔。迄今为止，TED在细胞系，神经胶质细胞和神经元在体外使用。 TED快递羧酸酯酶（CES）的基础上高效，重组目标高羧酸酯酶活性使用载体构建到内质网腔。最新的TED载体包含CES2红色荧光蛋白融合的核心要素，从而使两色同时成像。在ER中的游离钙离子的动态成像的一种颜色，而相应的ER​​结构以红色显示。的程序开始时，用慢病毒转导细胞。随后，受感染的细胞重新接种于盖玻片终于使活细胞成像的。然后，活细胞培养与乙酰甲酯（AM -酯）的形式，低亲和力的Ca 2 +指标，例如，MAG-Fluo4-AM Fluo5N-AM或MAG Fura2的调幅。在ER内酶切关闭从AM的Ca 2 +指示剂和亲水性荧光染料/ Ca 2 +的复合物形式的疏水性侧链的酯酶活性的内质网腔中形成并聚集。染料吸附量后，将细胞在一个倒置的共聚焦激光扫描显微镜分析。细胞连续灌流林格类似的解决方案和ER钙动力学直接可视化的时间推移成像。从内质网的钙离子的释放是确定的感兴趣区域的荧光强度下降，ER钙库的再填充而产生的荧光强度的增加。最后，荧光强度随时间的变化ΔF/ F 0通过计算确定。

该协议提供了一种非破坏性的方法，免费的ER钙的直接成像。低亲和性的合成的Ca 2 +释放指标被困于内质网腔的帮助下，一个对象的ER，重组酯酶的酶的活性。改进ER钙库动态加载的Ca 2 +指示剂染料的内质网腔，实现了直接和快速成像。 TED的细胞类型该方法的可行性已被证明在细胞系BHK21，HEK293 34，HeLa细胞17，SH-SY5Y，星形胶?...

Watch this Scientific Journal Video about Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED at JoVE.com

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

有针对性的酯酶诱导染料吸附量（TED）支持通过荧光成像分析细胞内钙库动态。该方法的基础上针对重组羧酸酯酶的内质网（ER），在那里它提高了合成的低亲和性钙本地揭露<sup&gt; +</sup&gt;指示剂染料在ER腔。

直接成像诱导ER钙有针对性的酯酶染料装载（TED）

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

direct-imaging-er-calcium-with-targeted-esterase-induced-dye-loading

Research

JoVE Journal

Biology

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

19.1K 次观看.  University of Wuerzburg. 有针对性的酯酶诱导染料吸附量（TED）支持通过荧光成像分析细胞内钙库动态。该方法的基础上针对重组羧酸酯酶的内质网（ER），在那里它提高了合成的低亲和性钙本地揭露 +指示剂染料在ER腔。

视频: Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

载入果蝇与神经末梢钙指标

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

科研

生物学

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

在体内2光子层钙成像对小鼠的2 / 3

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

Calcium Imaging of Cortical Neurons using Fura-2 AM

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium indicator dyes, which are BAPTA-based organic molecules that change their spectral properties in response to the binding of Ca2+ ions. Calcium indicator dyes fall into two categories, ratio-metric dyes like Fura-2 and Indo-1 and single-wavelength dyes like Fluo-4. Ratio-metric dyes change either their excitation or their emission spectra in response to calcium, allowing the concentration of intracellular calcium to be determined from the ratio of fluorescence emission or excitation at distinct wavelengths.  The main advantage of using ratio-metric dyes over single wavelength probes is that the ratio signal is independent of the dye concentration, illumination intensity, and optical path length allowing the concentration of intracellular calcium to be determined independently of these artifacts. One of the most common calcium indicators is Fura-2, which has an emission peak at 505 nM and changes its excitation peak from 340 nm to 380 nm in response to calcium binding.  Here we describe the use of Fura-2 to measure intracellular calcium elevations in neurons and other excitable cells.

Calcium signals play a key role in many cellular processes including gene expression, survival and differentiation. Here we demonstrate how to perform calcium imaging using Fura-2 AM. Calcium imaging is a valuable tool to study the regulation of intracellular calcium in real time and its regulation of signaling cascades.

使用FURA - 2上午皮层神经元的钙成像

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

大会，加载和分析超速离心机样品池对齐

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

活细胞钙成像相结合的siRNA介导的基因沉默标识CA 2 +泄漏通道内质网膜和监管机制

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

Expression and Purification of Virus-like Particles for Vaccination

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased biosafety and stability over use of live pathogens. Non-infectious and self-assembling, VLPs are used to present structural proteins as immunogens, bypassing the need for live pathogens or recombinant viral vectors for antigen delivery. In this article, we demonstrate the different stages of VLP design and development for future applications in preclinical animal testing. The procedure includes the following stages: selection of antigen, expression of antigen in cell line of choice, purification of VLPs/SVPs, and quantification for antigen dosing. We demonstrate use of both mammalian and insect cell lines for expression of our antigens and demonstrate how methodologies differ in yield. The methodology presented may apply to a variety of pathogens and can be achieved by substituting the antigens with immunogenic structural proteins of the user&#39;s microorganism of interest. VLPs and SVPs assist with antigen characterization and selection of the best vaccine candidates.

Here, we present a protocol for synthesizing virus-like particles using either baculovirus or mammalian expression systems, and ultracentrifugation purification. This highly customizable approach is used to identify viral antigens as vaccine targets in a safe and flexible manner.

表达和疫苗病毒样颗粒的纯化

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased ...

Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

Intracellular calcium (Ca2+) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of intracellular calcium release are the major intracellular calcium storage organelles, the endoplasmic reticulum (ER) and the more specialized sarcoplasmic reticulum (SR) in muscle cells. The dynamic release of calcium from these organelles is mediated by the ryanodine receptor (RyR) and the inositol 1,4,5-triphosphate receptor (IP3R) with refilling occurring through the sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. A genetically encoded calcium sensor (GECI) called CatchER was created to monitor the rapid calcium release from the ER/SR. Here, the detailed protocols for the transfection and expression of the improved, ER/SR-targeted GECI CatchER+ in HEK293 and C2C12 cells and its application in monitoring IP3R, RyR, and SERCA pump-mediated calcium transients in HEK293 cells using fluorescence microscopy is outlined. The receptor agonist or inhibitor of choice is dispersed in the chamber solution and the intensity changes are recorded in real time. With this method, a decrease in ER calcium is seen with RyR activation with 4-chloro-m-cresol (4-cmc), the indirect activation of IP3R with adenosine triphosphate (ATP), and inhibition of the SERCA pump with cyclopiazonic acid (CPA). We also discuss protocols for determining the in situ Kd and quantifying basal [Ca2+] in C2C12 cells. In summary, these protocols, used in conjunction with CatchER+, can elicit receptor mediated calcium release from the ER with future application in studying ER/SR calcium related pathologies.

Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

We present protocols for the application of our targeted genetically-encoded calcium indicator (GECI) CatchER+ for monitoring rapid calcium transients in the endoplasmic/sarcoplasmic reticulum (ER/SR) of HEK293 and skeletal muscle C2C12 cells using real-time fluorescence microscopy. A protocol for the in situ Kd measurement and calibration is also discussed.

监测ER / SR钙释放与目标Ca<sup&gt; 2+</sup&gt;传感器捕捉器<sup&gt; +</sup

Intracellular calcium (Ca2+) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of ...

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

The overall goal of this methodology is to give the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform 2D dual color dSTORM images of microtubules and intermediate filaments in fixed cells

2维直接随机光学重建显微成像中间丝和微管

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, ...

Fluorescence Recovery after Photobleaching of Yellow Fluorescent Protein Tagged p62 in Aggresome-like Induced Structures

Fluorescence recovery after photobleaching (FRAP)&#160;is a microscopy technique that can be used to quantify protein mobility in live cells. In a typical FRAP experiment, steady-state fluorescence is observed by repeated imaging with low-intensity laser light. Subsequently, the fluorescent molecules are rapidly and irreversibly impaired via brief exposure to high-intensity laser light. Information about protein mobility is obtained by monitoring the recovery of fluorescence. We used FRAP to determine the mobility of p62 in aggresome-like induced structures (ALIS) in murine macrophages after stimulation with lipopolysaccharide (LPS). Because many existing FRAP protocols are either incomplete or complex, our goal was to provide a comprehensive, practical, and straightforward step-by-step protocol for FRAP experiments with live cells. Here, we describe RAW264.7 macrophage transfection with yellow fluorescent protein-p62 (YFP-p62), induction of ALIS by exposing the cells to LPS, and a step-by-step method for collecting prebleach and postbleach FRAP images and data analysis. Finally, we discuss important factors to consider when conducting a FRAP experiment.

We describe a comprehensive and practical protocol for fluorescence recovery after photobleaching experiments with live cells. Although the protocol was used to measure the mobility of yellow fluorescent protein-tagged p62 in aggresome-like induced structures, it can be applied to a variety of microscopy systems and fluorescent proteins.

黄荧光蛋白标记 p62 在攻击样诱导结构中光漂白后的荧光恢复

Fluorescence recovery after photobleaching (FRAP)&#160;is a microscopy technique that can be used to quantify protein mobility in live cells. In a typical ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

小鼠胰腺胰岛细胞亚群中的钙动力学成像

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...