这项工作是支持部分由美国国家癌症研究所P30 CA23074，美国癌症协会的机构研究资助74-001-31，和大学。亚利桑那胃肠孢子（NCI / NIH的CA9506O）。

1。 S2细胞显微镜前的准备工作施耐德S2细胞均来自俄勒冈ř 果蝇的胰酶消化后期胚胎。施耐德公司的原始文化类型的细胞的混合物，但变得更均匀继续通过1。他们已被描述为类似血细胞的基因表达巨噬细胞样（他们是吞噬）。 S2细胞可从ATCC，DGRC，或Invitrogen公司。 A.生长介质的选择多家媒体已用于文化S2细胞。媒体没有提及需要5％的CO 2的气氛。 <ol><li>施耐德最初使用的10％热灭活胎牛血清（FBS），她的定义介质（施耐德的果蝇培养基）的补充。虽然胎牛血清比下面提到的无血清培养基介质中较昂贵的，它具有相对较低的自体荧光等epifluorescence显微镜活细胞的一个不错的选择。但是，它含有足够的金属，以刺激诱导金属硫蛋白启动子（例如，到Invitrogen的PMT载体亚克隆基因）的控制下的这些转基因的表达水平低，造成“漏”方可上岗基因的表达。 </li><li>市售一些无血清培养基（如Sf900二，Hyclone公司SFX - 昆虫和昆虫随心）。这些媒体是相对便宜，但使用金属硫蛋白启动子调控的转基因相比，施耐德公司的中期，通常是刺激较高水平的表达。此外，这些媒体相比，Schneiders“中/ FBS（一个重要的考虑进行活细胞荧光显微镜）时，产生显着的自体荧光。 </li><li>或者，抗生素可添加到中期，这些通常是青霉素G和硫酸链霉素（50-100单位/ ml和50-100微克/毫升的终浓度分别）。此外，抗真菌剂，两性霉素B，可以增加至250纳克/毫升（终浓度）。 </li></ol> B.文化条件 S2细胞易于维护，在正常的实验室温度和大气条件。当成像活延长时间的S2细胞，细胞死亡的首要原因是脱水和光毒性。脱水是很容易预防，保持足够的介质，在显微镜上的菜。光毒性是一个棘手的问题，需要最大限度地减少细胞的累积暴露到高强度，高能量的光，而成像经常捕捉到足够的空间和时间分辨率的有趣事件。 <ol><li> S2细胞生长在组织培养塑料通常为圆形和松散的附着。聚合细胞生长致密的单层，那么，越来越多，更多的细胞将起飞和成长中的悬浮。对于固定和活体细胞的显微镜，成像有很大的提高，如果细胞诱导的盖玻片上，扁平化。这一招（2。A. 2。下文所述）。 </li></ol> 2。 S2细胞的显微镜 A.活细胞显微镜我们用倒置显微镜观察生活镀上玻璃底菜的S2细胞。下文所述的玻璃底菜将保持在一个条件非常相似，他们的正常培养条件的细胞，还允许活细胞，通过显微镜高档玻璃检查。 <ol><li>制备玻璃底菜 组件： <ol class="lalpha"><li> Sylgard 184有机硅弹性体套件 这是胶水将盖玻片的菜。该套件有两个部分，树脂基组件和固化剂，使用前的重量比是10:1（树脂：固化剂）混合。 要准备树脂/固化剂混合物： <ol class="lroman"><li>称取大约5克的树脂塑料，重量船。注意树脂的重量。 </li><li>计算固化剂是由树脂重量乘以0.1需要多少。这种固化剂添加量相同的权衡船使用新的传输吸管。 </li><li>搅拌彻底混合组件。不要担心出现的泡沫，他们会慢慢消失。 </li><li>现在已经准备好胶水。它具有蜂蜜般的一致性，将至少一个小时的可用。 5.5克胶水足以使大约75菜，但是这取决于如何吝啬你当你申请的胶水。 </li></ol></li><li>塑胶35毫米培养皿 我们不使用手机的文化品位菜，因为我们只是在细胞附着到玻璃底部（而不是塑料）感兴趣 - 所以我们用更便宜，标准的培养皿。一些显微镜阶段圆形夹具容纳35mm培养皿;如果你有这个，确保你的菜适合钳。令人惊讶的是，并非所有35毫米的菜肴都具有相同的外径。 </li><li>盖玻片 选择适合你的显微镜和需要的盖玻片。什么更便宜的散装玻璃盖（例如，VWR，22x22毫米方形玻璃从昂贵的特种玻璃，光蚀刻的网格（例如，电子显微镜学，23x23毫米的圆形，网格化，无2，猫没有。72264-23）没有。1.5，猫没有。48366-227）可以使用。注意：必须稍大玻璃（至少1mm左右），比孔，你会在培养皿中，并也必须小于直径的菜。如果你打算买方形玻璃，玻璃完全覆盖（延长一点点超越）圆孔，但不会达到双方的菜。如果你喜欢干净的玻璃盖，这样做之前，胶合他们的菜。 </li><li>电钻 我们用变速和一个锁定的按钮，可连续使用一个手钻。钻井菜是一台钻床要容易得多，但生产规模小玻璃底菜没有理由购买一台钻床。 </li><li>钻头 使用一个¾英寸铲钻头（实话实位在此网页上图为：http://en.wikipedia.org/wiki/Drill_bit）。当然，该位的直径决定盘底部的孔的直径。 </li><li>锥形磨钻头 与粗砂粒和一个直径约铁锹位相同，使用一个圆锥状的研磨位。所有的孔已钻后，使用此位柏迪走在从孔底部的菜，项目边缘的塑料碎片。塑料碎片上的菜的外表面，将坐在盘对防止冲洗的玻璃盖。不要担心粘到盘的内部的小塑料件：他们不会影响玻璃盖或细胞。 </li><li>发泡胶盒厚的墙壁 这是用作钻井表面。聚苯乙烯泡沫塑料密封胶带条框的外侧（我们使用1.5英寸的透明胶带封箱胶带）;封箱胶带，最大限度地减少发泡胶位，而钻进逃生。 </li></ol> 大会 <ol class="lroman"><li>将发泡胶盒的录音表面上一个35mm的培养皿底部的一半;地方的菜，使的它的底部是对发泡胶盒。按住两侧的菜坚决，通过菜钻了一个洞。注意：你的手指拿着菜，将会从一个旋转的钻头一英寸的一小部分。当然，这是很危险的。停钻，如果盘开始旋转。如果你有一台钻床和/或一个聪明的方法，牢牢按住菜，使用它们。我们从来没有发生事故，但做任何你需要的，使此过程中的安全。 </li><li>钻进你的所有菜肴后，顺利使用磨钻头的孔外边缘。我们地方的电钻，在其一侧位投影在柜台边。这次演习是沉重的东西，但柔韧举行（我们使用含沙10公斤的重型塑料袋）的地方。使用锁定按钮保持连续运行演练，然后顺利你的菜，每道菜和移动对磨削位抚平粗糙边缘的新洞的轮辋。 </li><li>所有的菜放在一个大烧杯底部，并用蒸馏水冲洗数次，除去小，松散件的塑料或泡沫塑料（将浮动）。之后，躺在盘底部，取出晾干。 </li><li>当菜干，准备好你的Sylgard 184胶水。套用一个小圆圈上的每道菜的外表面钻孔周围的胶水。没有太多的胶水是需要的，事实上，太多的胶水将一个烂摊子。孔放在一个玻璃盖，在接触胶水。玻璃盖应该坐下顺利，均匀地对菜。如果胶水不完全孔周围分布，则可能是胶之间的玻璃盖和盘底部缺少小的差距。最初不用担心这些差距;通常胶水将这些差距蠕变，以填补他们。 </li><li>所有的玻璃盖粘合后，回去检查，但没有被胶水封闭缺口的菜肴。在任何的差距，适用于少量的胶水民建联的差距附近的玻璃盖的边缘;胶水将灯芯的差距。 </li><li>预留设置的菜，底部，晾干。这可能需要在室温下的天或更长时间，但你可以提高固化速度，在一个温暖的孵化器的菜肴。 </li><li>如果您需要无菌的菜，放盘底部和在组织培养罩盖和传播出来。不要把盘底部的盖子。相反，转动件，使他们的内表面是可怕的ctly面临的紫外线灯泡。杀菌的紫外线灯打开至少45分钟。如果您计划在错那外套的菜（见下文），不消毒，直到他们都涂后的菜肴。 </li><li>现在准备使用的菜。 </li></ol></li><li>刀豆蛋白A 被发现的凝集素，刀豆蛋白A（ConA），刺激S2细胞表面涂有与ConA 3对扁平化。当S2细胞接种到涂与ConA盖玻片，他们的传播，并成为优秀的显微镜标本。 <ol><li>对ConA大衣纯盖玻片，扩散片顶部的封口膜的清洗盖玻片录制下来到台上。 0.5 mg / mL的溶液刀豆蛋白A（无菌水）每个盖玻片表面，然后放置10μLs遍布整个盖玻片顶部材料。干燥后，储存在清洁，干燥的容器中的盖玻片。镀膜玻璃可在组织培养罩的紫外灯消毒。 </li><li> ConA的大衣玻璃底菜，同样蔓延到上部10μLsConA的解决方案（即盖面）的玻璃。干燥后，可消毒的菜，如上面所述。 ConA涂层的菜肴或盖玻片，在室温下保存，可用于配制后的几个月。 </li></ol></li><li>成像<ol class="lalpha"><li>轻轻重悬的RNAi治疗的S2细胞，并将它们传送到一个​​ConA涂层的玻璃底菜，含2 MLS中等。健康的细胞接触的ConA涂层的盖玻片，将坚持严格，并开始蔓延。约15分钟，这个过程应该成为明显，而应该由45分钟至1小时完成。注意：贴壁细胞不能进行细胞分裂ConA的涂层表面由于其严密的附件，并会随着时间的推移多倍体。种子细胞在ConA涂层的菜肴将长时间保持健康，但不会在这些菜增殖。 </li><li>细胞附着后，中期可以交换，如果旧媒体autofluorescent。这可能不是需要使用小场深处目标的共焦或反卷积显微镜或宽视场显微镜的研究。 </li><li>由于S2细胞健康，然后在室温/正常的气氛，没有特殊的条件或设备时所需的细胞在显微镜。我们经常采取长的时间推移电影，生活S2细胞。在实践中，这些实验的运行时间是有限的漂白和光毒性的常见问题的。 </li></ol></li></ol> B.固定细胞显微镜<ol><li>玻璃底菜与普通盖玻片：其中之一时，可以使用固定细胞。当使用普通盖玻片，放入一个单独的35mm的菜（这并不需要组织文化品位）一个单一的ConA涂层的盖玻片，加2 MLS媒体，然后转移入菜的RNAi治疗的细胞。细胞盖玻片后，取出介质，简单地用一个适当的缓冲液（PBS效果很好）的细胞，并添加固定液（见下文）的菜。之后，可以去除盖玻片从盘和进一步处理，例如由免疫。 </li><li>固定：有多种方法已被用于修复S2细胞。最好的固定方法将决定如何以及固定液保留利益的功能，不破坏或丢失的抗原表位或标签蛋白。有些程序需要之前固定的提取步骤，以揭露某些蛋白质的抗原决定簇位于密集或结构紧凑。由于S2细胞的文学是相当广泛的，它通常是可以找到发布的方法来优化您的利益S2细胞结构修复。 <ol class="lalpha"><li>甲醇是一种常见的的固定液。甲醇应冷（至少-20℃），无水（[类型3A分子筛可以被添加到抽象的水甲醇）。我们寒意约300毫升的无水甲醇在有盖的防爆20 ° C冰箱1L玻璃烧杯。甲醇是冷的，当它从冰箱中取出，迅速取出介质从玻璃底菜（或菜含有盖玻片），和的菜，然后迅速投身到甲醇，让它有。甲醇的烧杯中，通常认为大约有十几家菜。迅速开展工作，并尽快返回烧杯（含菜）冰柜。固定是在10-15分钟内完成。之后，从烧杯中删除的菜，倒在他们剩余的甲醇，加入PBS / 0.1％Triton X - 100的的补充水分。他们现正准备为标准的染色程序。 </li><li>甲醛是另一种常见的固定液。我们使用10％的缓冲液中的甲醛（PBS是可以接受的，但使用最适合自己需要的缓冲区，）。我们使用这种甲醛的浓度相对较高，因为S2细胞缺乏细胞质否则就会帮助维持细胞形态和组织在固定过程中的中间丝。 </li><li>成废物烧杯中倒入固定液固定细胞，并用三个简短的PBST洗（PBS + 0.1％TritonX - 100）。 </li><li>接下来，添加阻止细胞15分钟的解决方案1毫升。我们经常使用PBST 5％正常山羊血清。 </li><li>倒掉阻塞的解决方案和直接添加到细胞抗体（封闭液稀释）。 100μL抗体溶液应完全覆盖细胞。回盘的Petri盖，以防止抗体溶液蒸发。让我们为30分钟，在室温下的细胞抗体孵育。 </li><li>取出抗体的解决方案和执行三个PBST洗。使用转移吸管加入2毫升PBST菜。等待5分钟之间每洗（无需漩涡菜）。 </li><li>倒掉最后的清洗，并添加100μL二次抗体（封闭液稀释），直接到细胞。回盘的Petri盖，以防止抗体溶液蒸发。让我们为30分钟，在室温下的细胞抗体孵育。 </li><li>删除辅助抗体和执行3个5分钟用PBST洗。倾销的最后一次洗涤后，加入您的安装媒体的选择直接到细胞几滴。我们使用0.1 M的丙基没食子酸溶解成甘油溶液：PBS（9:1），并存储在20 ° C。更换的Petri盖子，在黑暗的环境中存储的菜。 </li></ol></li></ol> 3。限制/问题任何实验系统，S2细胞的使用有局限性。首先，S2细胞培养通常表现出有丝分裂指数低（约1％的无血清培养基），而很多转化的哺乳动物细胞系有丝分裂指数高达10倍以上。因此，对于有丝分裂表型的研究，S2的细胞培养需要较长的搜索，找到适当上演细胞。二，细胞周期研究，药物治疗是非常有效的逮捕S2细胞，在特定的细胞周期。然而，化合物具有可逆的细胞周期逮捕在培养的哺乳动物细胞的影响不容易冲刷S2细胞。因此，对于同步S2细胞增殖的协议尚未确定。第三，S2细胞不迁徙，也不显示上皮特点。 4。预期结果 S2细胞中的最大的卖点是其作为一个模型系统的实用工具。他们评估你感兴趣的蛋白质的细胞功能特别有用：S2细胞进行治疗的RNAi使用的双链RNA，在实验室中合成，很容易（相对便宜），和他们成为活细胞分析和固定细胞免疫。此外，S2细胞中的一个关键的好处是，使他们能够保持在实验室中，使用价格相对低廉的无血清培养基和无组织培养孵化器的缓解。 一个典型的实验中，将涉及电镀S2细胞（比如，一个6或96孔板）在一个适当的组织培养容器，加入自制的双链RNA井，然后保持文化逐渐耗尽通过RNAi作为目标蛋白质。根据对目标蛋白质被耗尽，细胞作为靶蛋白击倒显示形态学和/或行为的改变。以减少目标蛋白表达到最低限度所需的时间是特定的蛋白质，并应通过Western印迹法确定。 通常情况下，S2细胞不坚持紧紧塑料或玻璃，而这将构成一个问题需要处理的细胞镜检的实验。然而，S2细胞可诱导广泛扁平化，电镀表面涂有与外源凝集素，刀豆蛋白A（ConA）。对ConA电镀一小时内，大部分细胞已失去了其作为一个广泛的传播对ConA的表面（图1）的结果圆润的外观。现正准备作进一步处理（例如，通过固定和免疫）或显微镜观察活细胞的细胞。 S2细胞中也可以转（使用各种化学试剂或电），可以瞬时表达的外源基因或稳定整合到基因组（从而创造一个维护部门经过反复的外源基因的细胞株）的基因。转染可以用于多种用途，如固定或活细胞（图2），或者说利益的标记结构在体内的下拉或免疫实验等为诱饵，在荧光标记蛋白的表达分析方法的依赖，凑注册结构工程师，生物实验正在处理的问题。一个常用的方法是分析的RNAi治疗的S2细胞固定细胞免疫荧光（图3）。例如，免疫通常是用果蝇基因库的全基因组屏幕，以迅速确定有趣的活动在体内的蛋白质。同时消耗S2细胞中的多个靶蛋白，以检查潜在功能靶蛋白之间的相互作用，分析，可以更详细的。和分析，可以延长到活细胞，观察RNAi的动态过程的影响。同样，S2细胞是这种类型的分析，特别是非常适合的，因为他们可以镀ConA涂层的玻璃底菜，倒置显微镜多小时的可视化，而不需要一个环境室。 <img src="/files/ftp_upload/1982/1982fig1.jpg" alt="图1" /> 图1。相位对比图像S2细胞后镀上刀豆蛋白A镀膜玻璃底部盘（20倍的放大倍率）。这些数字表明镀后的分钟。请注意，这些细胞成为阶段深色涂层的盖玻片上，因为它们拼合。细胞扁平化是15分钟（箭头）是明显的，基本上是60分钟完成。右面板：放大贴壁细胞的形象;的扩展和扁平利润都清晰可见（箭头）。比例尺（四个左侧面板），20微米。 <img src="/files/ftp_upload/1982/1982fig2.jpg" alt="图2" /> 图2。S2细胞瞬时转染与融合构建nucleophosmin（细胞核标记）融合的荧光，EGFP（绿色）组成。这些细胞被镀上ConA涂层的玻璃底的菜，然后与DIC和epifluorescence成像。比例尺，15微米。 <img src="/files/ftp_upload/1982/1982fig3.jpg" alt="图3" /> 图3固定S2细胞免疫染色工党（绿色;一个中心粒标记），微管（红色），赫斯特为染色体染色（蓝色）。前固定和免疫细胞受到一个为期4天的治疗，要么控制的RNA干扰或RNAi击倒慢性非传染性疾病，一个动蛋白样蛋白，促进纺锤体极“ 聚焦 ” 4。注意叉开在慢性非传染性疾病的RNAi治疗的细胞纺锤体两极和混乱的主轴。比例尺，2.5微米。

<ol>
	<li>Schneider, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lines+derived+from+late+embryonic+stages+of+Drosophila+melanogaster.">Cell lines derived from late embryonic stages of Drosophila melanogaster.</a> J Embryol Exp Morphol. 27, 353-365 (1972).</li><li>Rogers, S. L., Rogers, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+Drosophila+S2+cells+and+their+use+for+RNAi-mediated+loss-of-function+studies+and+immunofluorescence+microscopy.">Culture of Drosophila S2 cells and their use for RNAi-mediated loss-of-function studies and immunofluorescence microscopy.</a> Nat Protoc. 3, 606-611 (2008).</li><li>Rogers, S. L., Rogers, G. C., Sharp, D. J., Vale, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+EB1+is+important+for+proper+assembly,+dynamics,+and+positioning+of+the+mitotic+spindle.">Drosophila EB1 is important for proper assembly, dynamics, and positioning of the mitotic spindle.</a> J Cell Biol. 158, 873-884 (2002).</li><li>Goshima, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+required+for+mitotic+spindle+assembly+in+Drosophila+S2+cells.">Genes required for mitotic spindle assembly in Drosophila S2 cells.</a> Science. 316, 417-421 (2007).</li></ol>

对于细胞生物学领域中，一个理想的系统将保持成本低廉，易于操纵，并适合各种技巧。果蝇 S2细胞中满足这些要求，并让他们迅速成为越来越多的细胞的制度选择生物实验室。 我们提出了一个简要概述的方法，准备S2细胞显微镜。 S2细胞中的一个显着优点是他们成长，在正常的气氛和室温，因此，它们可以长时间没有任何特殊的维护要求的显微镜左。即使他们一般都...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>S2 cells</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>CRL-1963</td>
<td><a href="http://www.atcc.org/">http://www.atcc.org/</a></td>
</tr>
<tr>
<td>S2 cells</td>
<td>&nbsp;</td>
<td>DGRC</td>
<td>stock number 6</td>
<td><a href="https://dgrc.cgb.indiana.edu/">https://dgrc.cgb.indiana.edu/</a></td>
</tr>
<tr>
<td>S2 cells</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>R690-07</td>
<td>&nbsp;</td>
<tr>
<td>Sf900 II</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10902-096</td>
<td>serum-free medium</td>
</tr>
<tr>
<td>HyClone SFX-Insect</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>SH30278</td>
<td>serum-free medium</td>
</tr>
<tr>
<td>Insect-Xpress</td>
<td>&nbsp;</td>
<td>BioWhittaker</td>
<td>12-730</td>
<td>serum-free medium</td>
</tr>
<tr>
<td>Schneider's S2 medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11720-034</td>
<td>Add 10% (final) heat-inactivated FBS to make complete medium.</td>
</tr>
<tr>
<td>Fetal bovine serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10438026</td>
<td>heat inactivated</td>
</tr>
<tr>
<td>100x antibiotic solution</td>
<td>&nbsp;</td>
<td>MP Biomedicals</td>
<td>91674049</td>
<td>contains penicillin (10000 U/mL), streptomycin (10000 &mu;g/mL), and amphotericin B (25 &mu;g/mL)</td>
</tr>
<tr>
<td>Concanavalin A</td>
<td>&nbsp;</td>
<td>MP Biomedicals</td>
<td>195283</td>
<td>&nbsp;</td>
<tr>
<td>Formaldehyde</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>15714</td>
<td>32% solution</td>
</tr>
<tr>
<td>Methanol</td>
<td>&nbsp;</td>
<td>Mallinckrodt Baker</td>
<td>9049</td>
<td>anhydrous, ACS grade</td>
</tr>
<tr>
<td>Molecular sieves</td>
<td>&nbsp;</td>
<td>Acros Organics</td>
<td>19724</td>
<td>type 3A</td>
</tr>		</tbody>
		</table>
		</div>

preparation of drosophila s2 cells for light microscopy

理想的实验系统，将便宜和易于维护，适合多种技术，将通过大量的文献和基因组序列数据库支持。培养的果蝇 S2细胞中，脱离20-24 小时 1岁胚胎的产品，具备所有这些特性。因此，S2细胞的细胞过程，包括发现的基因编码过程中的分子成分或利益机制的分析非常非常适合。其效用是最负责任的S2细胞的特点是与它们保持缓和，以其精湛的双链（DS）介导的RNA干扰（RNAi）的敏感性，以及居住或固定的可追踪性荧光显微镜细胞。 S2细胞可以生长在各种媒体，包括一些便宜，市售的无血清培养基，充分定义，2 。此外，他们成长优化，并迅速在21-24 ° C，可在各种容器中培养。哺乳动物细胞不同，S2细胞并不需要监管的氛围，而是与正常空气，甚至可以在保持密封烧瓶。 在S2细胞中的RNAi缓解的补充，是能够很容易分析实验诱导的表型相或固定或活细胞的荧光显微镜。文化作为一个单一的单层S2细胞的生长，但不显示接触抑制。相反，细胞往往生长在茂密的文化殖民地。在密度低，S2文化生长在玻璃或组织培养处理的塑料圆而松散连接。然而，S2细胞的细胞学检查可大大提高，诱使他们拉平简要地培养他们广泛的外源凝集素，刀豆蛋白A（ConA）3表面涂。 S2细胞中也可以稳定转染与荧光标记的标记，标签结构或细胞器在活细胞或固定细胞的兴趣。因此，细胞的微观分析通常的情况是这样的：第一，S2细胞（可以拥有转基因表达标签标记）通过RNAi治疗，以消除靶蛋白（S）。 RNA干扰治疗时间可以调整，以便在蛋白质的差异转动力学和，以减少细胞的创伤/死亡，如果目标蛋白是很重要的生存能力。接下来，处理后的细胞被转移到一盘菜含有盖玻片预涂与ConA诱导细胞扩散，紧紧坚持以玻璃。最后，细胞成像与研究者的显微镜模式的选择。 S2细胞，为需要扩展可视化活细胞的研究，特别是良好的，因为这些细胞停留在室温和常压下的健康。

Watch this Scientific Journal Video about Preparation of Drosophila S2 cells for Light Microscopy at JoVE.com

Preparation of Drosophila S2 cells for Light Microscopy

果蝇施耐德（S2）细胞基因的发现和功能分析系统越来越受欢迎。我们的目标是描述这样一个日益重要的实验系统，使S2细胞的显微技术。

制备果蝇 S2细胞，光学显微镜

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature ...

preparation-of-drosophila-s2-cells-for-light-microscopy

Research

JoVE Journal

Biology

Preparation of Drosophila S2 cells for Light Microscopy

22.4K 次观看.  University of Arizona (UOA). 果蝇施耐德（S2）细胞基因的发现和功能分析系统越来越受欢迎。我们的目标是描述这样一个日益重要的实验系统，使S2细胞的显微技术。

视频: Preparation of Drosophila S2 cells for Light Microscopy

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

电子显微镜对胚胎的制备果蝇胚胎心管

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

科研

生物学

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific shapes. Drosophila tracheal terminal cells have become a powerful genetic model to identify and elucidate the roles of genes required for generating cellular morphologies. Terminal cells are a component of a branched tubular network, the tracheal system that functions to supply oxygen to internal tissues. Terminal cells are an excellent model for investigating questions of cell shape as they possess two distinct cellular architectures. First, terminal cells have an elaborate branched morphology, similar to complex neurons; second, terminal cell branches are formed as thin tubes and contain a membrane-bound intracellular lumen. Quantitative analysis of terminal cell branch number, branch organization and individual branch shape, can be used to provide information about the role of specific genetic mechanisms in the making of a branched cell. Analysis of tube formation in these cells can reveal conserved mechanisms of tubulogenesis common to other tubular networks, such as the vertebrate vasculature. Here we describe techniques that can be used to rapidly fix, image, and analyze both branching patterns and tube formation in terminal cells within Drosophila larvae. These techniques can be used to analyze terminal cells in wild-type and mutant animals, or genetic mosaics. Because of the high efficiency of this protocol, it is also well suited for genetic, RNAi-based, or drug screens in the Drosophila tracheal system.

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Here, we present a method for light microscopy analysis of tracheal terminal cells in Drosophila larvae. This method allows for quick examination of branch and lumen morphology in whole animals and would be useful for analysis of individual mutants or screens for mutations affecting terminal cell development.

考试<em&gt;果蝇</em幼虫气管终端细胞光镜

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific ...

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized microtubules. Organelles move along these processes by microtubule motors. Easy maintenance, high sensitivity to RNAi-mediated protein knock-down and efficient procedure for creating stable cell lines make Drosophila S2 cells an ideal model system to study cargo transport by live imaging. The results obtained with S2 cells can be further applied to a more physiologically relevant system: axonal transport in primary neurons cultured from dissociated Drosophila embryos. Cultured neurons grow long neurites filled with bundled microtubules, very similar to S2 processes. Like in S2 cells, organelles in cultured neurons can be visualized by either organelle-specific fluorescent dyes or by using fluorescent organelle markers encoded by DNA injected into early embryos or expressed in transgenic flies. Therefore, organelle transport can be easily recorded in neurons cultured on glass coverslips using living imaging. Here we describe procedures for culturing and visualizing cargo transport in Drosophila S2 cells and primary neurons. We believe that these protocols make both systems accessible for labs studying cargo transport.

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells and cultured neurons are great systems for imaging of motor-driven organelle transport in vivo. Here we describe detailed protocols for culturing both cell types, their imaging and analysis of transport.

细胞器运输的培养<em&gt;果蝇</em&gt;细胞：S2细胞系和原代神经元。

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

在造血干细胞和祖细胞利用共聚焦和多光子显微镜五荧光蛋白标记的活体克隆跟踪

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Generating a &#34;Humanized&#34; Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper distribution of the genetic material requires that sister kinetochores on every chromosome become bioriented by attaching to microtubules from opposite spindle poles before progressing into anaphase. However, erroneous, non-bioriented attachment states are common and cellular pathways exist to both detect and correct such attachments during cell division. The process by which improper kinetochore-microtubule interactions are destabilized is referred to as error correction. To study error correction in living cells, incorrect attachments are purposely generated via chemical inhibition of kinesin-5 motor, which leads to monopolar spindle assembly, and the transition from mal-orientation to biorientation is observed following drug washout. The large number of chromosomes in many model tissue culture cell types poses a challenge in observing individual error correction events. Drosophila S2 cells are better subjects for such studies as they possess as few as 4 pairs of chromosomes. However, small molecule kinesin-5 inhibitors are ineffective against Drosophila kinesin-5 (Klp61F). Here we describe how to build a Drosophila cell line that effectively replaces Klp61F with human kinesin-5, which renders the cells sensitive to pharmacological inhibition of the motor and suitable for use in the cell-based error correction assay.

Generating a &quot;Humanized&quot; Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

This protocol describes how to generate a Drosophila S2 cell line that is sensitive to small molecule inhibitors of kinesin-5. The use of these cells in a cell-based error correction assay is also outlined.

生成一个“人性化”<em&gt;果蝇</em&gt; S2细胞敏感的驱动蛋白-5的药理抑制

Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper ...

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

果蝇的制备及心功能体内使用光学相干显微镜成像纵向（OCM）

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

高压冷冻、微波辅助对比度增强和最小树脂嵌入在体积成像中的生物样品制备

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

The larvae of Drosophila melanogaster show obvious light-avoiding behavior during the foraging stage. Drosophila larval phototaxis can be used as a model to study animal avoidance behavior. This protocol introduces a light-spot assay to investigate larval phototactic behavior. The experimental set-up includes two main parts: a visual stimulation system that generates the light spot, and an infrared light-based imaging system that records the process of larval light avoidance. This assay allows tracking of the behavior of larva before entering, during encountering, and after leaving the light spot. Details of larval movement including deceleration, pause, head casting, and turning can be captured and analyzed using this method.

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

This protocol introduces a light-spot assay to investigate Drosophila larval phototactic behavior. In this assay, a light spot is generated as light stimulation, and the process of larval light avoidance is recorded by an infrared light-based imaging system.

光斑分析分析果蝇拉瓦尔光轴

The larvae of Drosophila melanogaster show obvious light-avoiding behavior during the foraging stage. Drosophila larval phototaxis can be used as a model ...

Cryo-Electron Microscopic Grid Preparation for Time-Resolved Studies using a Novel Robotic System, Spotiton

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental challenge of great interest to the field of cryo-electron microscopy (cryo-EM). A few methodological strategies have been developed that support these &#34;time-resolved&#34; studies, one of which, Spotiton-a novel robotic system-combines the dispensing of picoliter-sized sample droplets with precise temporal and spatial control. The time-resolved Spotiton workflow offers a uniquely efficient approach to interrogate early structural rearrangements from minimal sample volume. Fired from independently controlled piezoelectric dispensers, two samples land and rapidly mix on a nanowire EM grid as it plunges toward the cryogen. Potentially hundreds of grids can be prepared in rapid succession from only a few microliters of a sample. Here, a detailed step-by-step protocol of the operation of the Spotiton system is presented with a focus on troubleshooting specific problems that arise during grid preparation.

The protocol presented here describes the use of Spotiton, a novel robotic system, to deliver two samples of interest onto a self-wicking, nanowire grid that mix for a minimum of 90 ms prior to vitrification in liquid cryogen.