谢谢凯文Miyashiro特丽Schochet博士，电镀和维护细胞培养，细胞培养提供本文档中所包含的图片。此外，谢谢凯文Miyashiro，彼得巴克利博士，Tiina Pertiz ARNA程序的输入。这项工作的经费是由国家老龄问题研究所，国立精神卫生和人力资源实际上从宾夕法尼亚州联邦查找资金。

1。细胞培养我们的实验室使用下面的实验的主要神经元大鼠海马文化。下面介绍如何创建和维护这些细胞培养的银行家协议 9，当然，这些细胞培养技术和任何既定的方法排列的详尽量身定做，它提供了一个特定的实验室的具体需求持续健康细胞的供应将是一个合适的替代。 <ol><li>五蒸压，圆形，12毫米盖玻片被放置在一个35毫米的菜，并涂以80μg/ mL的聚- D -赖氨酸在硼酸盐缓冲液O / N（兆瓦70-150,000），水（细胞培养级）冲洗，然后涂硼酸盐缓冲液O / N为1微克/毫升层粘连蛋白，然后再冲洗水。吴媒体（与Earle的盐和葡萄糖（0.6％W / V）为辅GlutaMAX，青霉素（100 ü /毫升），链霉素（100微克/毫升），马血清（10％V / V）的MEM）是补充和菜存储在一个孵化器（5％的CO 2，在37 ° C） 。 PDL150/laminin服务作为一个孤立的神经细胞成长为一个单层基板。可涂在盖玻片电镀前两个星期。 </li><li>在电镀的日子，从胚胎第18天大鼠海马神经元的主要是镀吴媒体中加入1.5毫升100,000细胞/ mL悬液。电镀后的四到六小时，每35毫米的菜是用0.75μL5毫米胞嘧啶β- D - arabinofuranoside（ARA - C），以防止任何污染的神经胶质细胞的生长。二十四小时后，电镀媒体更改为纪念厄尔的盐和L -谷氨酰胺，0.6％W / V葡萄糖，丙酮酸钠1毫米，和B27的补充。细胞被允许使用任何实验前增长至少两个星期，让完整的开发过程。 </li></ol> 2。准备移液器注:对核糖核酸酶的皮肤，唾液，甚至呼吸酶降解RNA的RNase的主要来源。当务之急是从这个角度观察，无RNase的技术，使样品不会成为与RNase的污染，并随后降解的协议。这包括处理的样品和试剂时，总是戴着手套，从来没有超过样本或试剂，并使用新箱消毒枪头和管子。通常情况下，不专门指定用于RNA工作的移液器净化抹下来核糖核酸酶处理解决方案，如核糖核酸酶距离（分子生物制品）。然而，这些解决方案将抑制任何下游的酶反应，所以它也是重要的，以防止这些治疗你的样品污染。 <ol><li>高压灭菌1.5毫米外径（OD值）玻璃吸液管。 </li><li>使用微量拉马，拉移液管直径适当的电生理记录，这可能会有所不同根据你的车夫，长丝， 移液器 10孔尺寸是不是太重要，因为尖端细胞采集前将被打破。 </li><li>小心地保存在一个无尘的环境中的提示将保持不变（最好使用微量存储罐）移液器。 </li><li>要在受控制的方式打破的提示，一方面举行绷紧你的手指之间和一个kimwipe非常吸管尖轻轻刷整个文件的1至2次。开放应靶细胞的大小约为75-100％。调整此步骤，直到达到预期的效果。 </li></ol> 3。准备文化，管，显微镜<ol><li>准备所需数量的1.7 mL的Eppendorf管。如果是单细胞扩增，或任何其他的收获材料的存储解决方案中使用的材料，加入2μL的PBS，第一链缓冲。 </li><li>收获，您将需要与光镜装有一个显微一个40x的目标。软管领先远离持有人使用吸管持有人。油管贴上一个稳定的表面，以防止不必要的运动期间收集的吸管。在管端插入针与鲁尔锁连接，使一个1 mL注射器可以在用户之间的连接和改变。 </li><li>如果使用盖玻片，工作一次盖玻片。如果有必要，转让一个新的35毫米盘*含有PBS或媒体选择单一盖玻片。时间越长，细胞的孵化器，更不健康，他们将成为和mRNA的配置更会偏离一个健康细胞。它是保存在孵化，在不与他们合作的其他盖玻片上的细胞的健康至关重要。尽快与孵化器以外的细胞。 </li></ol> *随着我们的设置是必要的，实际菜的墙壁是用35毫米盘的盖子，因为太高，允许对coversli适当的微进步第 4。收获细胞<ol><li>安全的微量微量持有。贴上微量持有人的micromanipulators插入。设置的40倍，目标。 </li><li>将菜在显微镜舞台上找到一个适合收获的细胞区域。在初级神经元文化的情况下，细胞从它的邻国应相对隔离，以防止周围的细胞和/或进程的收获。 SOMA和流程之间的mRNA转录的人口变化，如果在体mRNA的感兴趣只，护理应采取措施防止污染与流程SOMA。将你想收获的细胞在视野的中心。 </li><li>使用显微走向菜光路的解决方案的微量。降低到解决方案的枪头。应用正压从这个角度上，轻轻吹通过注射器。看看通过目镜。移液器应该出现在视野的阴影。远高于飞机的细胞，调整枪头的位置，直到它吸管尖粗的聚焦旋钮上的观点和重点领域内的。在这一点上，它应分摊的吸管孔的大小，如果过大或过小。实践收获会提供的能力，以确定正确的孔径大小，如果在这一点上已经取得。 </li><li>现在，提前向细胞的平面吸管。它是非常重要的提示是没有先进的太匆忙，造成它打破你想收集该地区从。为了防止这种情况，使用粗的重点，以推进推进对使用micromanipulators细胞枪头前焦平面。当你接近的细胞，用优良的焦点，直到细胞和枪头重点。停止应用正压之前到达飞机的细胞，以防止吹盖玻片。 </li><li>使用micromanipulators位置枪头，因此，它是触摸你想收获的细胞SOMA。 </li><li>使用注射器，适用于经口轻轻吸气​​，待细胞进入枪头。如果细胞是不能进入的吸管，轻轻地尖接近细胞和向下移动，直到它从盖玻片升降机。 </li></ol> 5。保存细胞<ol><li>使用显微操作，立即采取行动解决方案的吸管和。 </li><li>删除从持有人的吸管。 </li><li>一方面保持1.7 mL Eppendorf管中，并轻轻地打破移液器管的底部侧面提示。 （0.1毫升大关的目标。） </li><li>断尖为中心的管内，插入到顶端开口的吸管连接1-3毫升注射器针头。迅速压低柱塞迫使移液器的解决方案，喷入管。细胞应该留在最顶端的吸管和正确驱逐细胞，这应该是足够的。要小心不要接触任何液体管双方的提示毛细作用把吸管的液体倒回。 </li><li>快速降速使用的是台式离心管的内容，并立即冻结（储存于-80 ° C），或作进一步处理（最好）的冰地。 </li></ol> 6。 ARNA扩增<ol><li>第1轮第一链cDNA合成: 创建的mRNA / cDNA的杂交。 <ol><li>对于每一个单细胞采集量的5μL，添加下列1X收集管（冰）。反应可缩放收集量较大（2X 10μL收集卷等）。收集管加10-20％乘以下列试剂移液错误的帐户创建一个主结构。 ARNA ampification过程中的每一个后续反应。 <ul><li>在ICE </li><li> 1.2μL，dNTP的（2.5毫米） </li><li> 2.4μL，5倍的第一链缓冲</li><li> 0.3μLT7 -寡核苷酸（dT）引物（100纳克/μL） </li><li> 1.2μL的数码地面电视（100毫米） </li></ul></li><li>把音量高达10.25μL与核酸自由水。移液器组合和旋转简要使用离心。 </li><li>孵育5分钟，在70 ° C变性任何的mRNA的二级结构。立即置于冰上至少5分钟。 </li><li>添加（再次使用预混）: <ul><li> 0.3μL，RNasin（40 U /μL） </li><li> 0.45μL标三（200 U /μL） </li><li> 1μL的核酸游离水</li></ul></li><li>移液器组合和旋转简要。孵育1小时42 ° C。 </li><li>在70 ° C下孵育15分钟灭活上标。继续进行下一步。 </li></ol></li><li>第1轮第二链cDNA合成 创建从mRNA部分的mRNA / DNA杂交的RNA引物，以帮助合成Øf双双链cDNA。 <ol><li><ul><li>在ICE </li><li> 12μL第一链反应，地址: </li><li> 8μL的核酸游离水</li><li> 7.5μL，5倍的第二链缓冲</li><li> 0.75μLdNTP混合液（2.5毫米） </li><li> 0.25μL，DNA连接酶（10 U /μL） </li><li> 1μL，DNA聚合酶I（10 U /μL） </li><li> 0.25μL，核糖核酸酶H（2 U /μL） </li></ul>吹打调匀，和旋转简要。孵育2小时，在16 ° C。 </li><li>地址:1μLT4 DNA聚合酶（5 U /μL）。混合吹打和自旋简要。在16 ° C孵育10多分钟</li><li>清理反应使用的Qiagen公司MinElute试剂盒，为制造商的指示稍作修改 :11与500μL洗涤缓冲液750μL1次洗2次。洗脱核酸自由水。 </li><li>集中到2-4微升使用Speed​​vac或乙醇沉淀。乙醇沉淀，糖原作为一种核酸的载体和沉淀少量的DNA或RNA时使用。钠醋酸钠有助于中和负电荷的DNA骨干，帮助降水。这是没有必要让一个常规降水的主组合。 <ul><li>加入29μLDEPC水</li><li> 2μL，糖原（5mg/mL） </li><li> 1 / 10的体积（3μL）3M醋酸钠</li><li> 2.5卷（〜250μL）的100％冷乙醇</li></ul>拌匀。沉淀于-80℃（30分钟O / N）。 </li><li>离心20分钟，在4 ° C。 </li><li>去除上清，用800μL70％乙醇洗涤沉淀。请务必完全逐出吹打或振荡，以帮助清除多余的盐分沉淀。 </li><li>离心20分钟，在4 ° C。取出上清，空气干燥15-20分钟。 </li><li>悬浮在4μL核酸自由水。贮存于-20℃或-80 ° C或继续下一步。 </li></ol></li><li>回合在体外转录（IVT），1: 综合纳入到双链cDNA T7启动子的反义RNA。 <ol><li>这种反应是执行每除一个，而不是一个20μL的反应10μL，规模的制造商的说明使用的Ambion公司MEGAscript T7套件。12因此，以装配在室温下这种反应和以保持室温在组装过程中的缓冲区。提供的缓冲区，将沉淀的DNA，如果它是冰冷的。冰在不使用时保持NTPS和酶混合。 <ul><li> AT室温</li><li>双链的悬浮基因转移到薄壁PCR管。 </li><li> 4μL的NTP混合（18.75毫米） </li><li> 1μL10X反应缓冲液</li><li> 1μL，10倍的酶混合物</li></ul></li><li> 14个小时在37 ° C的一个热循环仪（最好）或孵化器。 </li><li>这种反应可以使用两种不同的方法使用Speed​​vac或乙醇沉淀的样品浓度清理。对于使用一个工具包清理，进行A.清洁与一个标准的酚/氯仿抽提方法，进行方法B </li><li>方法A: <ol type="a"><li>清理反应使用Ambion公司Megaclear套件，为制造商的说明了一些修改 13:洗2次，与500μL洗涤缓冲液750μL1次。洗脱步骤，添加50μL的核酸自由水中心之列，在70 ° C孵育10分钟。 10,000克为1分钟旋转。在一个新的管重复。合并洗脱液。 2-4μL使用Speed​​vac或乙醇沉淀浓缩样品。 </li><li>乙醇沉淀:醋酸铵在这种情况下，醋酸钠的地方使用，因为它是有效地防止游离核苷酸与核酸共同沉淀。这是很重要的，因为高浓度的NTP公司在IVT中的反应，它可以抑制下游反应使用。 <ul><li>加入50μLDEPC水</li><li> 2μL，糖原（20毫克/毫升） </li><li> 0.6卷（37.2μL），5M醋酸铵</li><li> 2.5卷（〜180μL），冷乙醇</li></ul></li><li>沉淀于-80℃（30分钟O / N）* </li><li>离心20分钟，在4 ° C。 </li><li>取出上清液，沉淀用800μL70％乙醇（核酸免费水）。一定要充分打跑的沉淀，去除所有多余的盐分。 </li><li>离心20分钟，在4 ° C。 </li><li>除去上清，空气干燥15-20分钟。 </li><li> 4μL的核酸自由水的悬浮颗粒。 </li></ol></li><li>方法B: <ol type="a"><li>另外，ARNA可以清理一个标准的酚/氯仿抽提。 <ul><li>加入50μLDEPC水</li><li> 2μL，糖原（20毫克/毫升） </li><li> 0.6卷（37.2微升）5M醋酸铵</li><li> 1卷（98μL），酚:氯仿:异戊醇25:24:1（平衡pH值7.8-8.0） </li></ul></li><li>涡为15秒。一半在表顶部的离心的最大速度为1.5分钟，离心。顶部的水层转移到一个新的试管中，2.5倍体积的冷乙醇（245μL）。 </li><li>沉淀于-80℃（30分钟O / N）。 </li><li>离心20分钟，在4 ° C。 </li><li> 800μL70％乙醇（核酸游离水），取出上清液和洗涤沉淀。一定要充分打跑的沉淀，去除所有多余的盐分。 </li><li>离心20分钟，在4 ° C。 </li><li>除去上清，空气干燥15-20分钟。 </li><li> 4μL的核酸自由水的悬浮颗粒。 *样品，可能会冻结在此温度下在一夜之间孵化。它已被证明，这实际上可以增加产量核酸。 </li></ol></li></ol><li>第2轮第一链cDNA合成<ol><li><ul><li>在ICE </li><li>添加1μL，随机引物（0.05毫克/毫升）* </li></ul></li><li>热火在70 ° C 10分钟。立即置于冰上至少5分钟。 </li><li><ul><li>添加2μL，5倍的第一链缓冲</li><li> 1μL的数码地面电视（100毫米） </li><li> 0.5μL，dNTP的（2.5毫米） </li><li> 0.5μL，RNasin（40 U /μL） </li><li> 1μL标三（200 U /μL） </li></ul></li><li>吹打调匀，和旋转简要。允许在室温下坐了10分钟。这一步是必需的，允许延长短的随机引物，逆转录反应之前进行。 </li><li>孵育30分钟，在42 ° C。 </li><li>加热5分钟在95 ° C至变性DNA / RNA杂交的RNA。置于冰上至少5分钟。储存在-20 ° C或-80 ° C或立即继续下一步。 *随机引物的浓度是重要的。如果浓度过高，该产品将每一轮的放大截断。 </li></ol></li><li>第2轮第二链cDNA合成<ol><li><ul><li>在ICE </li><li>添加2μL，T7，以oligo（dT）引物（10毫微克/μL）* </li></ul></li><li>在70 ° C 5分钟热立即置于冰上至少5分钟。自旋简要。 </li><li><ul><li>新增43.5μL的核酸自由水</li><li> 15μL的5倍第二链缓冲</li><li> 1.5μLdNTP混合液（2.5毫米） </li><li> 2μL，DNA聚合酶I（10 U /μL） </li></ul></li><li>吹打调匀，和旋转简要。孵育2小时，在16 ° C。 </li><li>加入2μLT4 DNA聚合酶（5 U /μL） </li><li>吹打调匀，和旋转简要。在16 ° C孵育10多分钟</li><li>清理在6.2.3节中使用的Qiagen公司Minelute试剂盒的反应。 </li><li>在第6.2.4至6.2.8与Speed​​vac或乙醇沉淀，浓缩到2-4μL。 *注引物T7寡糖不同的股权集中度（DT）相比，第一轮第二链cDNA合成。 </li></ol></li><li>第2轮在体外转录 6.3节中演出。 <ol><li>重复第6.4至6.6倍所需的数字。注意，每一轮ARNA产品在较短的扩增结果的后续。轮扩增的数量应限于这个原因。通常情况下，放大两到三个回合足够的放大，从一个单一的细胞进行基因芯片分析收获的材料。在微阵列分析的情况下，第三轮IVT反应是为制造商的指示使用Illumina公司TotalPrep RNA扩增试剂盒。此过程纳入ARNA检测微阵列芯片上，然后将其使用的生物素标记的UTP。 </li><li>第三轮ARNA纳米RNA试剂盒使用一个Nanodrop或安捷伦生物分析仪测量的浓度。 </li></ol></li></li></ol> 7。代表性的成果在不到2分钟就可以完成从初级神经元文化的一个单细胞收获成功，取决于性向（见图1）。然而，收获的时间会有所不同系统之间和干预实验操作。单细胞受到ARNA程序（见图4A及4B）在总放大ARNA微克金额的结果，并产生特有的宽峰分析与生物分析仪（见图3）。三个回合就可以完成了至少3天，允许单细胞基因表达的快速分析。 <img src="/files/ftp_upload/2634/2634fig1.jpg" alt="图1" /> 图1所示是一个一个孤立的神经元的收获成功的一个例子。我们塞莱反恐执行局的一个相对较低的密度区域（一）向所需的细胞（B）和先进的枪头。第三个图像（c）显示，已经收获后的细胞领域。需要注意的是周围的进程保持盖玻片。 <img src="/files/ftp_upload/2634/2634fig2.jpg" alt="图2" /> 图2所示的两幅图像枪头，这是一个有效的收获不恰当的大小。这些技巧会导致不完整的收获（A）和周围环境（b）分别收获。 <img src="/files/ftp_upload/2634/2634fig3.jpg" alt="图3" /> 图3。以下收获和使用一个生物分析仪的放大，分析，建议检查扩增RNA的分布和数量。一个从单细胞的物质成功扩增产量低微克的总金额，将有一个平稳和广泛的分布。 <img src="/files/ftp_upload/2634/2634fig4.jpg" alt="图4" /> 第一轮（A）和（二）ARNA程序的第二轮的原理图图4所示。

<ol>
	<li>Davis, J. E., Eberwine, J. H., Hinkle, D. A., Marciano, P. G., Meaney, D. F., McIntonsh, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodological+considerations+regarding+single-cell+gene+expression+profiling+for+brain+injury.">Methodological considerations regarding single-cell gene expression profiling for brain injury.</a> Neurochemical Research. 29, 1113-1121 (2004).</li><li>Eberwine, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+molecular+biology.">Single-cell molecular biology.</a> Nature Neuroscience. 4, 1155-1156 (2001).</li><li>Kamme, F., Salunga, R., Yu, J., Tran, D., Zhu, J., Luo, J., Bittner, A., Guo, H., Miller, N., Wan, J., Erlander, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+microarray+analysis+in+hippocampus+CA1:+Demonstration+and+Validation+of+Cellular+Heterogeneity.">Single-cell microarray analysis in hippocampus CA1: Demonstration and Validation of Cellular Heterogeneity.</a> The Journal of Neuroscience. 23, 3607-3607 (2003).</li><li>Hinkle, D., Glanzer, J., Sarabi, A., Pajunen, T., Zielinski, J., Belt, B., Miyashiro, K., McIntosh, T., Eberwine, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+neurons+as+experimental+systems+in+molecular+biology.">Single neurons as experimental systems in molecular biology.</a> Progress in Neurobiology. 72, 129-142 (2004).</li><li>Ginsberg, S. D., Elarova, I., Ruben, M., Tan, F., Counts, S. E., Eberwine, J. H., Trojanowski, J. Q., Hemby, S. E., Mufson, E. J., Che, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+gene+expression+analysis:+Implications+for+Neurodegenerative+and+Neuropsychiatric+Disorders.">Single-cell gene expression analysis: Implications for Neurodegenerative and Neuropsychiatric Disorders.</a> Neeurochemical Research. 29, 1053-1064 (2004).</li><li>Eberwine, J., Spencer, K., Miyashirto, K., Mackler, S., Finnell, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complementary+DNA+synthesis+in+situ:+methods+and+applications.">Complementary DNA synthesis in situ: methods and applications.</a> Methods Enxymol. 216, 80-100 (1992).</li><li>Eberwine, J., Yeh, H., Miyashiro, K., Cao, Y., Nair, S., Finnell, R., Zettel, M., Coleman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+gene+expression+in+single+live+neurons.">Analysis of gene expression in single live neurons.</a> Proc. Natl. Acad. Sci. USA. 89, 3010-3014 (1992).</li><li>Kelz, M. B., Dent, G. W., Therianos, S., Marciano, P. G., McIntosh, T. K., Coleman, P. D., Eberwine, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+antisense+RNA+amplification+and+microarray+analysis+as+a+tool+for+studying+neurological+degeneration+and+restoration.">Single-cell antisense RNA amplification and microarray analysis as a tool for studying neurological degeneration and restoration.</a> Sci. Aging Knowledge Eviron. 1, re1-re1 (2002).</li><li>Banker, G. A., Cowan, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+hippocampal+neurons+in+dispersed+cell+culture.">Rat hippocampal neurons in dispersed cell culture.</a> Brain Research. 126, 397-425 (1977).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> P-1000 &#38; P-97 Pipette Cookbook. , (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> MinElute Handbook For MinElute Reaction Cleanup Kit. , 28-29 (2004).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Megascript kit. , 7-8 .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> MEGAclear kit. , 4-5 .</li><li>Eberwine, J., Kacharmina, J. E., Andrews, C., Miyashiro, K., McIntosh, T., Becker, K., Barrett, T., Hinkle, D., Dent, G., Marciano, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+expression+analysis+of+tissue+sections+and+single+cells.">mRNA expression analysis of tissue sections and single cells.</a> The Journal of Neuroscience. 21, 8310-8314 (2001).</li><li>Van Gelder, R. N., von Zastrow, M. E., Yool, A., Dement, W. C., Barchas, J. D., Eberwine, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplified+RNA+synthesized+from+limited+quantities+of+heterogeneous+cDNA.">Amplified RNA synthesized from limited quantities of heterogeneous cDNA.</a> Proc. Natl. Acad. Sci. USA. 87, 1663-1667 (1990).</li><li>Miyashiro, K., Dichter, M., Eberwine, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+nature+and+differential+distribution+of+mRNAs+in+hippocampal+neuritis:+Implications+for+neuronal+functioning.">On the nature and differential distribution of mRNAs in hippocampal neuritis: Implications for neuronal functioning.</a> Proc. Natl. Acad. Sci. USA. 91, 10800-10804 (1994).</li><li>Sul, J., Wo, C. K., Zeng, F., Jochems, J., Lee, M. T., Kim, T. K., Peritz, T., Buckley, P., Cappelleri, D. J., Maronski, M., Kim, M., Kumar, V., Meaney, D., Kim, J., Eberwine, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+transfer+produces+a+predictable+cellular+phenotype.">Transcriptional transfer produces a predictable cellular phenotype.</a> Proc. Natl. Acad. Sci. USA. 106, 7624-7629 (2009).</li><li>Sow, F. B., Gallup, J. M., Sacco, R. E., Ackerman, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+revisited+as+a+tool+for+transcriptomic+analysis:+Application+of+an+excel-based+qPCR+preparation+software+(PREXCEL-Q).">Laser capture microdissection revisited as a tool for transcriptomic analysis: Application of an excel-based qPCR preparation software (PREXCEL-Q).</a> Int. J. Biomed. Sci. 5, (2010).</li><li>Vandewoestyne, M., Deforce, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+in+forensic+research:+a+review.">Laser capture microdissection in forensic research: a review.</a> Int J Legal Med. 124, (2010).</li><li>Eberwine, J., Bartfai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+transcriptomics+of+hypothalamic+warm+sensitive+neurons+that+control+core+body+temperature+and+fever+response+Signaling+asymmetry+and+an+extension+of+chemical+neuroanatomy.">Single cell transcriptomics of hypothalamic warm sensitive neurons that control core body temperature and fever response Signaling asymmetry and an extension of chemical neuroanatomy.</a> Pharmacol. Ther. , (2010).</li></ol>

Eberwine博士是ARNA Eberwine博士是股份持有人的定位技术已经被授权的专利的发明者。

注意事项和故障排除<ul><li>您可能需要采取前后靶细胞的分离，显示的图像和其余半影是原封不动。 </li><li>如果太多的解决办法是进入你正在收获的吸管，可以使用3路鲁尔锁配件和举行正压管路中的注射器的柱塞。所需音量会有所不同的处理类型，但对于大多数应用程序应罚款高达5μL。 </li></ul>分子生物学技术的一般技巧<ul><li>涡所有股票管和旋转下来之前加入反应。从不涡酶。 </li><li>充分混合反应，然后再添加酶，以确保该缓冲区是在适当的浓度。 </li><li>如果可能的话使用stratacooler保持在-20 ° C的酶的股票。否则，删除权在使用前，保持在冰上，并立即返回到-20 ° C </li><li>务必掌握混合时，准备一个以上的反应，以减少移液错误。计算所需的样本数量的基础上的量和添加10-20％。 </li><li>商店RNA在-80 ° C至延缓退化。 RNA是最稳定的存储在缓冲区来延缓的RNA apurination在酸性条件下发生的。 </li></ul> ARNA程序的一般纲要图4A说明ARNA程序的第一轮。在第一链反应的T7以oligo（dT）引物聚- T的部分选择绑定的多聚腺苷酸尾巴mRNA的物种（长的白色矩形）。一些小分子RNA是聚腺苷酸化和将捕获此过程。然而，更重要的是，在细胞中最丰富的RNA，核糖​​体RNA，不会。这寡核苷酸逆转录引物的行为，使用的mRNA为模板合成的基因的互补链（长的灰色矩形）。 T7的T7以oligo（dT）引物部分采用T7 RNA聚合酶启动子，开始mRNA的反义序列帧。这是用于在体外转录反应。 接下来，在前面的步骤中创建的mRNA / DNA杂交的mRNA部分是由核糖核酸酶制造的RNA"引物"（白色的小矩形）冈崎片段，在滞后链DNA的合成中创建类似H水解。 DNA聚合酶I总理DNA合成的DNA互补的mRNA作为模板使用的RNA片段。当它到达下一个RNA片段，其5&#39;到3&#39;核酸酶活性，消除了核苷酸和替换脱氧核糖核酸。 DNA连接酶添加到结扎任何股的龙头股的更换是不完整的的。 T4 DNA聚合酶是填加所在地区的RNA片段作为最初的引物为我创造的DNA聚合酶担任双重之前执行IVT的反应，然后将其纯化的双链cDNA钝端。 在IVT反应，T7 RNA聚合酶结合并入的双重链cDNA合成的反义RNA分子的正义链为模板（长的黑色矩形）的T7启动子。这可作为扩增步骤中，数以千计的反义RNA分子是从每一个双链cDNA的分子（图4A）生产。 与逆转录反应以来的起始RNA的第一轮稍有不同，如图4B所示，第二轮开始反义（固体黑色矩形），缺乏聚腺苷酸化尾，由T7 -寡核苷酸的目标（dT）引物，在第一轮。因此，这种反应是引物与随机引物（小的灰色矩形）和核糖核酸（RNA）随后变性。第二链反应的T7以oligo（dT）引物，这在前面的逆转录反应创造了义RNA的3&#39;端结合聚一个序列，然后催芽。另一个IVT反应是在第一轮以同样的方式进行。第二轮通常至少重复一次，实现了从一个单细胞的三轮放大。 应用可以转化为大量的应用，我们已经在本文介绍的技术。单细胞分离协议可以修改用于急性切片14虽然在技术上更具挑战性，同样的原则适用于在这个交替的准备。此外，如果吸管的大小略有调整，细胞生理学的录音可以收获前允许的生理输出背后的分子机制以及控制调查。另一个稍微修改是从细胞胞体分离的过程。对于这个应用程序 15个，收集细胞机构之一吸管，然后返回与一个新鲜的吸管，收集100-300确定dendritES或轴突每收集管16。 一旦细胞已被收割，mRNA的丰度和化学成分的比较可以之间的不同，即使在相同的细胞群。纳入第三轮ARNA生物素化的双绞线，允许芯片的分析，以确定这些相对表达丰度。原mRNA的人口的构成也可以ARNA使用下一代测序的过程后才能确定。放大ARNA也可以用来确定细胞表型转化研究，在其中一种细胞类型的mRNA的全套是转成不同的细胞类型，以促使前者，后者的细胞类型的表型的转变， TIPeR已知的实验室开发的一个过程 [17]这些研究为研究疾病状态和细胞表型等研究特别有用，目前在实验室进行。可以进行RT - PCR或定量PCR扩增材料，以确认细胞特异性基因的表达。此外，转染或转导效率的评价，可在单细胞水平上。 优点和局限性抽象表示，隔离消除了单细胞分析分析异质细胞群的平均作用。这些平均影响歪曲过较丰富的成绩单和平均和预防许多低丰度的转录检测单个细胞内mRNA的丰度。流式细胞仪可用于单个细胞进行排序，但这种方法需要了解细胞的特异性标志物和昂贵的设备 。18激光捕获显微切割使用紫外线或红外线激光捕获显微切割系统，使单细胞甚至亚细胞捕获，但需要利益的细胞坐落在非常薄的部分表面。19类似的流式细胞仪，激光捕获显微切割也需要昂贵的设备。 上文所述的电极收集技术的主要好处之一是，宝贵的电生理数据，可从细胞收获前的兴趣，允许在同一细胞进行功能和转录分析20的缺点是我们的技术它需要使用micromanipulators的经验。熟悉调查与micromanipulators会发现这个技术非常直观的，然而，没有这样的经验，个人需要成为舒适与所需的精细动作。 在任何扩增技术所固有的某些成绩单优惠放大，根据大小和核苷酸组成。4,6,7聚合酶链反应（PCR）为基础的技术，如逆转录聚合酶链反应（RT - PCR检测）和cDNA快速扩增结束（赛）的结果成绩单指数扩增，而ARNA放大线性放大过程的结果。因此，ARNA程序的主要优点之一在于在其能力，更好地维护只能由线性放大反对成倍放大，在放大过程中发生的任何错误或偏见的相对丰度的mRNA转录错误和偏见。 ARNA过程，再加上微阵列分析，可以比较相似或不同的形态，处理或未经处理的单细胞之间的mRNA的丰度。此外，扩增材料可以提交给下一代测序5,8然而，护理应在这样的分析以来，相反的程序使用的随机引物，寡DT催芽过程描述上述偏见的放大3&#39;端的mRNA和一般结果在随后扩增每轮材料略有缩短。应谨慎分析芯片的结果与标准方法，为一些虚假的缺席的电话，可能会出现从略有缩短放大材料。此外，同时测序结果的确会提供完整的5&#39;最原始的mRNA序列，可能会错过一些基因的5&#39;序列。基于这些原因，轮扩增的数量应该是有限的。

A correction was made to <a href="http://http://jove.unicartagenaproxy.elogim.com/video/2634/transcriptome-analysis-of-single-cells">Transcriptome Analysis of Single Cells</a>. There were errors in the volumes used in the aRNA Amplification section.

Erratum: Transcriptome Analysis of Single Cells

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Spiegelgas coverslips</td>
<td>&nbsp;</td>
<td>Carolina Biological</td>
<td>63-3029</td>
<td>&nbsp;</td>
<tr>
<td>Nunc 35x10 mm culture dishes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>12-565-90</td>
<td>&nbsp;</td>
<tr>
<td>Water for cell culture</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>17-724Q</td>
<td>For making solutions used for cell culture and rinsing coverslips</td>
</tr>
<tr>
<td>Poly-D-Lysine MW70-150K</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>6407</td>
<td>&nbsp;</td>
<tr>
<td>Laminin, ultrapure</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>354239</td>
<td>&nbsp;</td>
<tr>
<td>Boric acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B0252</td>
<td>&nbsp;</td>
<tr>
<td>MEM with Earle&#x2019;s salts and glutamax</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>41090-101</td>
<td>For plating cells</td>
</tr>
<tr>
<td>D-glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G8769</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Horse serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16050</td>
<td>&nbsp;</td>
<tr>
<td>Cytosine beta-D-arabinofuranoside</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C1768</td>
<td>&nbsp;</td>
<tr>
<td>MEM with Earle&#x2019;s salts and L-glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11095-098</td>
<td>For growing cells</td>
</tr>
<tr>
<td>Sodium pyruvate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P2256</td>
<td>&nbsp;</td>
<tr>
<td>B27 serum-free supplement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>Borosilicate glass capillary tubes (1.5mm O.D, 100mm length)</td>
<td>&nbsp;</td>
<td>Kimble Chase</td>
<td>34500 99</td>
<td>Any borosilicate glass pipette will work as long as the proper bore size is attained. </td>
</tr>
<tr>
<td>HBSS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14175</td>
<td>Any solution will work as long as the components won&#x2019;t interfere with any future processing (i.e. no Ca2+ or Mg2+)</td>
</tr>
<tr>
<td>1ml syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309628</td>
<td>&nbsp;</td>
<tr>
<td>Needle</td>
<td>&nbsp;</td>
<td>BD</td>
<td>&nbsp;</td>
<td>Gauge depends on the diameter of the pipettes</td>
</tr>
<tr>
<td>dNTP mix</td>
<td>&nbsp;</td>
<td>Amersham</td>
<td>28-4065-51</td>
<td>&nbsp;</td>
<tr>
<td>dt-T7 oligo</td>
<td>&nbsp;</td>
<td>Custom</td>
<td>Midland Certified</td>
<td>&nbsp;</td>
<tr>
<td>Second strand buffer (5x)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>Y01129</td>
<td>&nbsp;</td>
<tr>
<td>DTT</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Supplied with second strand buffer</td>
</tr>
<tr>
<td>E.coli DNA Ligase</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>100002324</td>
<td>&nbsp;</td>
<tr>
<td>DNA polymerase I</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>100004926</td>
<td>&nbsp;</td>
<tr>
<td>Rnase H</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>18021-071</td>
<td>&nbsp;</td>
<tr>
<td>Megascript T7 kit</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AM1334</td>
<td>&nbsp;</td>
<tr>
<td>Random primers</td>
<td>&nbsp;</td>
<td>BMB</td>
<td>11034731001</td>
<td>&nbsp;</td>
<tr>
<td>Superscript III Reverse Transcriptase</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>56575</td>
<td>in kit (18080-044) comes with first strand buffer (Y02321) and DTT</td>
</tr>
<tr>
<td>MEGAclear Kit</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>1908</td>
<td>&nbsp;</td>
<tr>
<td>MinElute Reaction Cleanup Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>28206</td>
<td>&nbsp;</td>
<tr>
<td>T4 DNA polymerase</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>100004994</td>
<td>&nbsp;</td>
<tr>
<td>Rnasin</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>N251B</td>
<td>&nbsp;</td>
<tr>
<td>Illumina TotalPrep RNA Amplification Kit</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AMIL1791</td>
<td>&nbsp;</td>
<tr>
<td>Flaming/Brown micropipette puller</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>P-87</td>
<td>Sutter has many other models, many are discussed in the cookbook</td>
</tr>
<tr>
<td>Micromanipulator</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>Many other micromanipulators will work such as the newer Eppendorf models</td>
</tr>
<tr>
<td>Pipette Holder</td>
<td>&nbsp;</td>
<td>Warner Instruments</td>
<td>MP-S15A</td>
<td>Will vary with micromanipulator and pipette O.D.</td>
</tr>
<tr>
<td>Bioanalyzer RNA 6000 Nano Kit</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>5067-1511</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

transcriptome analysis of single cells

许多基因表达分析技术，依赖于从组织匀浆或细胞培养细胞的异质种群隔离材料 。1,2,3大脑的情况下，如海马区包含一个复杂的安排不同类型的细胞，每个不同的mRNA的概况。收获的单细胞的能力，可以在进入细胞群之间和国家内部的分子差异的深入调查。我们描述了一个简单，快速的方法收获​​细胞作进一步处理。中电经常使用的移液器利用隔离（使用共同愿望）的利益的一个单元格，并方便地存入一个Eppendorf管中，它与任意数量的分子生物学技术的进一步处理。我们的协议可以被修改为树突细胞培养或急性片甚至单个细胞的收获。 我们还描述了作为一个主要的下游应用单细胞隔离ARNA扩增法。这种方法是开发以前，我们的实验室作为替代其他基因的表达分析技术，如逆转录或实时聚合酶链反应（PCR）。4,5,6,7,8这种技术提供的线性放大聚腺苷酸化的RNA开始只有毫微微克的物质，并造成微克的反义RNA。线性放大的材料，提供了一个比PCR分离细胞转录的组成部分的相对丰度指数扩增更准确的估计。基本程序包括两轮扩增。简单地说，一个T7 RNA聚合酶启动子网站纳入双链从mRNA转录的cDNA。一夜之间在体外转录（IVT），反应，然后执行其中T7 RNA聚合酶产生许多从双双链cDNA反义转录。第二轮重复这一过程，但自起始原料，一些技术上的差异，是反义RNA。它是标准的重复第二轮，三轮扩增。通常情况下，第三轮在体外转录反应是用生物素标记的核苷三磷酸，使反义RNA可以杂交和检测到一个芯片上。 7,8

Watch this Scientific Journal Video about Transcriptome Analysis of Single Cells at JoVE.com

Transcriptome Analysis of Single Cells

在这篇文章中，我们描述了一个简单的方法从大鼠原代神经元的文化和随后的转录组学分析使用ARNA扩增单细胞的收获。这种方法推广到任何类型的细胞。

单细胞的转录组学分析

Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in ...

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

29.7K 次观看.  University of Pennsylvania. 在这篇文章中，我们描述了一个简单的方法从大鼠原代神经元的文化和随后的转录组学分析使用ARNA扩增单细胞的收获。这种方法推广到任何类型的细胞。

视频: Transcriptome Analysis of Single Cells

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

科研

神经科学

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

阈上的神经活动的单细胞和单穗的分辨率的光学的方法记录

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

连续光漂白划定单雪旺细胞在神经肌肉接头处

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

标记的单细胞在中枢神经系统<em&gt;果蝇</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

激光捕获显微切割的丰富种群的创伤性脑损伤后神经元或单个神经元的基因表达分析

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Peering at Brain Polysomes with Atomic Force Microscopy

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, formed by ribosomes, mRNAs, several proteins and non-coding RNAs, represent integrated platforms where translational controls take place. However, while the ribosome has been widely studied, the organization of polysomes is still lacking comprehensive understanding. Thus much effort is required in order to elucidate polysome organization and any novel mechanism of translational control that may be embedded. Atomic force microscopy (AFM) is a type of scanning probe microscopy that allows the acquisition of 3D images at nanoscale resolution. Compared to electron microscopy (EM) techniques, one of the main advantages of AFM is that it can acquire thousands of images both in air and in solution, enabling the sample to be maintained under near physiological conditions without any need for staining and fixing procedures. Here, a detailed protocol for the accurate purification of polysomes from mouse brain and their deposition on mica substrates is described. This protocol enables polysome imaging in air and liquid with AFM and their reconstruction as three-dimensional objects. Complementary to cryo-electron microscopy (cryo-EM), the proposed method can be conveniently used for systematically analyzing polysomes and studying their organization.

While ribosome structure has been extensively characterized, the organization of polysomes is still understudied. To overcome this lack of knowledge, we present here a detailed preparation protocol for accurate imaging of mammalian polysomes by atomic force microscopy (AFM) in air and liquid.

在脑核糖凝视与原子力显微镜

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, ...

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

视网膜神经节细胞定义子集的单细胞RNA-Seq

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

应用流式细胞仪分析单侧坐骨神经和背根神经节的免疫细胞

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

成人脊髓细胞核在大规模平行单核 RNA 测序中的分离

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...

Analysis of Spliceosomal snRNA Localization in Human Hela Cells Using Microinjection

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment called the Cajal body. However, sequences that direct snRNA localization into this subnuclear structure have not been known until recently. To determine sequences important for accumulation of snRNAs in Cajal bodies, we employed microinjection of fluorescently labelled snRNAs followed by their localization inside cells. First, we prepared snRNA deletion mutants, synthesized DNA templates for in vitro transcription and transcribed snRNAs in the presence of UTP coupled with Alexa488. Labelled snRNAs were mixed with 70 kDa-Dextran conjugated with TRITC, and microinjected to the nucleus or the cytoplasm of human HeLa cells. Cells were incubated for 1 h and fixed and the Cajal body marker coilin was visualized by indirect immunofluorescence, while snRNAs and dextran, which serves as a marker of nuclear or cytoplasmic injection, were observed directly using a fluorescence microscope. This method allows for efficient and rapid testing of how various sequences influence RNA localization inside cells. Here, we show the importance of the Sm-binding sequence for efficient localization of snRNAs into the Cajal body.

Biogenesis of spliceosomal snRNAs is a complex process involving various cellular compartments. Here, we employed microinjection of fluorescently labelled snRNAs in order to monitor their transport inside the cell.

使用微注射在人赫拉细胞中分体血球定位分析

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment ...