Name: isolating intestinal stem cells from adult drosophila midguts by facs to study stem cell behavior during aging
Uploaded: 2014-12-16T15:00:00.000+00:00
Duration: 10 min 57 s
Description: Watch this Scientific Journal Video about Isolating Intestinal Stem Cells from Adult Drosophila Midguts by FACS to Study Stem Cell Behavior During Aging at JoVE.com

We are grateful to Gabriele Allies for excellent technical assistance. We thank the University Ulm Medical Faculty for the use of the FACS Core Facility and the Institut f&#252;r Molekulare und Zellul&#228;re Anatomie for using the confocal microscope. We thank S. Hayashi for the esg-Gal4, UAS-GFP fly line. This project is funded by the Federal Ministry of Education and Research (BMBF, Forschungskern SyStaR). A.T. is supported by SFB 1074 (Project A2). A.T. and G.A. are supported by the Deutsche Forschungsgemeinschaft (DFG, FE578/3-1). H.M.T. is a member of the International Graduate School in Molecular Medicine Ulm (GSC 270).

注意:如果该协议被用于在第一时间通过FAC分选以分离ISC的，下面的控制是必需的，以便最初正确设置的FACS参数:解离的细胞的野生型（ 例如，瓦特1118）果蝇肠而不SYTOX。游离细胞从野生型（ 如W 1118），果蝇肠与SYTOX（见步骤3.6）。游离的细胞，而不SYTOX的ESG -Gal4，UAS-GFP飞线肠。 1.准备解决方案和餐具肠道夹层<ol><li>准备4-6小瓶每片含40雌蝇从ESG -Gal4，UAS-GFP飞线。 </li><li>从10X PBS原液制成500毫升之1X PBS（1.8毫米的NaH 2 PO 4·H 2 O，8.4毫米的Na 2 HPO 4·2H 2 O，175毫米氯化钠，调节pH值至7.4）。 </li><li>准备一个3.5在1×PBS中％琼脂糖溶液（3.5克电泳级琼脂糖在100毫升1×PBS）中。通过倾倒该溶液到培养皿（直径8.5厘米），以覆盖底部制备夹层板中。琼脂糖层防止在清扫过程中损坏钳的提示。后的凝胶凝固存储夹层板在4℃下。 </li><li>制备300毫升1×PBS + 1％BSA中的新鲜解剖前，并将瓶在冰上或4℃。 </li><li>打开离心机，并让它冷却至4℃。 </li><li>火焰2巴斯德玻璃吸管的提示，以平滑的边缘。 </li><li>清洁两对镊子和一个刀片，用70％的乙醇。 </li><li>放置四到六个1.5ml微量管中，用于收集解剖肠在冰上。 </li></ol> 2.制备中肠的胃肠道和夹层<ol><li>麻醉苍蝇从一个小瓶（40苍蝇）与二氧化碳对标飞床，全部斩首苍蝇用刀片，并将它们转移到解剖盘。倾冷的1×PBS / 1％BSA溶液进入解剖盘以覆盖琼脂糖凝胶。苍蝇会浮。 </li><li>抢飞腹部与一对钳子的，同时保持胸部与另一对钳子。分离从腹部胸部。肠道将是可见的和前肠/作物将最有可能仍然被连接到胸部。 </li><li>抢肠道和拉出来的胸部。展开肠道，抓住作物和拉肠向前稍微远离腹部。 </li><li>抓住腹部的后端与一对镊子和向前打开角质层与另一对钳子的边缘。要小心，不要破坏突出肠组织。拉后端远离打破角质层并继续拉向后非常平缓，直到整个肠道被拉出腹腔。作物可能必须事先除去，如果它太大，以适应通过体腔。 </li><li>取下前肠，马氏管，后肠和卵巢留下光秃秃的肠。 </li><li>使用玻璃巴斯德吸管，批处理解剖肠的转移到含有冷的1×PBS / 1％BSA溶液1.5 ml离心管中，并保持该管在冰上。样品必须在2小时处理。 </li><li>经过整批的解剖完成后，冲洗清扫菜用双蒸水洗掉遗留的碎片。开始解剖下一批肠的（重复步骤2.1-2.7）。在2个小时的解剖尽可能多批地，然后继续执行步骤3.1。 </li></ol> 3.消化肠道组织，以收获细胞FAC的排序<ol><li>除去从肠中的1×PBS / 1％BSA溶液，加入500微升0.5％的胰蛋白酶-EDTA溶液，以每个样品。 </li><li>涡流以及20秒，并培育样品通过温和的摇摆/在20rpm旋转在室温下25-30分钟。 </li><li>涡旋后，再次约30分钟，让完整肠组织沉到试管底部。小心删除的悬浮用火烧玻璃巴斯德吸管的细胞，并通过35微米尼龙过滤它们网格到一个新的1.5 ml离心管。 </li><li>旋转细胞向下，在100×g离心5分钟，在4℃。值得注意的是，在开始的时候摘要，一个细胞团可能不会在第一次看到，但将随着组织摘要进步明显。 <ol><li>胰蛋白酶溶液仔细转移回含剩余完整肠组织的原始样品管中。避免颗粒转移太多细胞。 </li><li>轻轻重新悬浮于400μl冷的1×PBS / 1％BSA中的细胞沉淀。保持含在冰上离解的细胞的离心管，并用铝箔覆盖的管，以保护细胞免于光。 </li><li>旋涡再次含有剩余完整肠组织的胰蛋白酶溶液，然后将样品到摇杆另外30分钟，在室温下进行。 </li></ol></li><li>重复步骤3.3到3.4.3，直到所有的肠组织已被消化。从所有离心管结合的解离的细胞到一个离心管中。这可能需要一个离心步骤在3.4，因为所有的细胞悬浮液合并可能超过1.5毫升的体积。保持细胞悬浮液在冰上并保护细胞免受光。 </li><li>旋离解的细胞向下，在100×g离心5分钟，在4℃。小心除去尽可能多留下大约50-100微升的微量离心管中的1×PBS / 1％BSA溶液的上清作为可能的。轻轻悬浮在800微升的1×PBS / 1％BSA中含有SYTOX（1:20,000）离解的细胞。 </li><li>通过用细胞过滤网卡扣帽（35微米尼龙网）过滤细胞转移细胞悬液至5毫升圆底Falcon管中。始终保持细胞样本的冰和避光。细胞现在已经准备好进行排序。 </li><li>吸取600微升的RNAlater溶液到每个微量离心管到其中的细胞将被排序为后续的RNA分离。对于其他的下游应用的细胞可以被收集在不同的解决方案， 例如在无菌PBS中。 </li></ol> 4. FAC排序来隔离肠道干细胞<ol><li>交换机上的流式细胞仪至少一小时之前排序。确保流体系统是无气泡。 </li><li>选择70微米的喷嘴尺寸，以将细胞注入到鞘流体流。 </li><li>调整芯流体流的振幅，使得（使用70微米的喷嘴时6-7）的间隙值对应的参考值。让我们开始进行排序之前，核心流体流稳定。 </li><li>按照这个顺序，当最初设定的参数进行排序: <ol><li>加载从野生型的胆量得到了游离细胞。首先，调整FSC和SSC电压绘制在散点图中心的细胞。其次，调整FITC（绿色荧光蛋白）的电压，使所有的细胞被绘制低于10 2上的对数x轴。设置FITC参数设置限制自发荧光。 </li><li>加载加入从野生型胆与SYTOX得到的解离的细胞。调整从死，SYTOX阳性细胞太平洋蓝与FSC-A散点图太平洋蓝电压值和门来识别和分离的活细胞。 </li><li>装载从ESG -Gal4，UAS-GFP的飞线而不SYTOX得到的解离的细胞，并调整在FITC电压，以确保所有的GFP阳性细胞的散点图中绘出。 </li></ol></li><li>加载从ESG -Gal4获得的分离细胞，用SYTOX UAS-GFP飞线补充说。调整从死，SYTOX阳性细胞（门P1）的太平洋蓝电压值和门来识别和分离的活细胞。 <ol><li>将SSC-A和FSC-A门识别GFP-阳性细胞（基于直方图），通过大小和粒度（栅极P2）来确定。 </li><li>将FSC-H和FSC-W门来识别和排除基于它们的大小（门P3）GFP阳性细胞双峰。 </li><li>将SSC-H和SSC-W门来识别和排除根据他们的粒度（门P4）GFP阳性细胞双峰。 </li><li>使用门的P4描绘GFP阳性细胞在直方图显示细胞（计数）相对于GFP强度的数目。 GFP阳性细胞的两个截然不同的峰值将是可见的。创建每个峰（门P5和P6）的栅极。栅极P5包含富集对ISC的，并且可以从细胞中栅极P6分别排序的细胞群。 </li><li>背栅中的等高线图，以验证该两个不同的GFP阳性细胞群含有活细胞（与栅极P1比较），并正确大小的细胞（具有栅极的P2进行比较）。 </li></ol></li><li>将细胞分类到包含600＆＃1.5 ml离心管181;升RNAlater中的解决方案。排序完成以低流速（最大2.0，70微米的喷嘴，1000次/秒，70磅），并采用一个双向纯度排序。 </li><li>排序完成后，立即涡选的细胞短暂，并保持在冰上的微量离心管中，直到在继续进行RNA分离或其它下游应用。 </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+expression+profiling+from+FACS-isolated+cells+of+the+Drosophila+intestine.">RNA expression profiling from FACS-isolated cells of the Drosophila intestine.</a> Curr. Protoc. Stem Cell Biol. 13 (27), (2013).</li></ol>

The authors declare that they have no competing financial interests.

所提出的协议描述了一种方法，从老少成年果蝇中肠，随后其可用于进一步的分子分析如下一代测序隔离ISC的。从ESG -Gal4 GFP阳性细胞群的FAC分选，UAS-GFP飞线已经由若干组21,22来实现的。然而，到现在为止的GFP阳性细胞的两个不同的峰的存在已被要么忽视或低估。我们表明，这种分离不仅代表的细胞类型（ISCS和EBS）的两个不同的群体，也表明GFP阳性细胞的两个峰留在老化过程中明显地分开的。这种看法是高度相关的，有价值的研究人员感兴趣的老化过程研究的干细胞或祖细胞。从旧肠孤立综合监控系统已经阻碍了事实，没有分子标记结合，以确定ISC的中老年人肠。唯一的标志，美nambiguously标识的旧肠ISC的是磷酸histone3（PH3），因为ISC的是唯一的分裂细胞在肠12,13。然而，PH 3是不合适的标记物以分离ISC的自除以干细胞在任何给定时间点的数目太低，以便获得ISC的体面量用于随后的分析。飞线ESG -Gal4，UAS-GFP是一种用于研究人员谁研究ISC功能，维护和分化中最常见的飞线。我们对ISC的动态平衡的分子调节的现有知识是基于研究中，特定的分子和信号通路已被操纵（在7中综述）。因此，我们仍然缺乏知识老化过程中发生的内源性分子变化。在本文中所描述的方法封闭该间隙，并基于这样的事实，ISC的可以基于从在老化过程中的任何时间点成人肠它们的大小，粒度和GFP强度进行分离。 而建立和优化该方法，我们已经发现以下的步骤是一个可靠的结果是至关重要的。约200胆需要为了获得ISC的量好为FAC分选被解剖。夹层的速度是至关重要的，取决于个人的做法。一个训练有素的人可以在20-30分钟剖析40胆量。由于GFP也表示在ESG -Gal4的马氏管，UAS-GFP飞线，就必须从肠完全删除马氏管。解剖肠必须保存在阴凉环境，避免组织退化和减少组织RNA酶的活性。因此，解剖肠必须从解剖盘转移到含有最多20-30分钟后冷的1×PBS / 1％BSA溶液的微量离心管并保持在冰上。然而，解剖肠不应启动组织离解，用胰蛋白酶之前置于冰上超过2小时。最effici耳鼻喉科的做法是解剖苍蝇尽可能多批次内的这2个小时，然后开始胰蛋白酶消化这些批次。如果需要更多的肠，它们可以在胰蛋白酶消化的孵育时间进行解剖。 尽管胰蛋白酶是一种非常有效的酶，并可能对细胞有害的影响，但我们仍然获得足够的生活，健康的细胞用于后续FAC分选。我们的程序的关键步骤，其允许完整细胞的分离是离解的细胞从胰蛋白酶溶液，每30分钟除去。如果解剖肠孵育2个半小时，含在室温下的解决方案胰蛋白酶，我们观察到死，SYTOX阳性细胞增加了20-30％。应当指出，我们使用了胰蛋白酶溶液含有EDTA中。 EDTA存在可能是通过螯合所需的细胞外基质分子的正常功能的钙离子和镁离子促进组织崩解。 </P&gt; 最关键的步骤，以成功地排序ISC的从老少胆是FACS和使用所描述的控制和跟随一个特定的排序等级门控参数的适当的初始设置。我们进行了FAC上ARIA II流式细胞仪（FACSDiva软件）排序。重要的是，在仪器被至少一小时接通之前分拣热身的激光器。值得注意的是，当第一次被执行的ISC的FAC分选，所述参数用于排序和补偿需要被使用上述控制设置:（1）从野生型解离的细胞（ 例如，瓦特1118）果蝇肠不添加SYTOX的 - 设置此参​​数（步骤4.4.1）防止基于其自身荧光分拣细胞; （2）分离的细胞从野生型（ 如W 1118），果蝇肠与SYTOX加-设置此参数（步骤4.4.2）允许从活细胞中分离死（死细胞具有高的自体荧光和会污染排序GFP阳性细胞）; （3）从ESG -Gal4的肠细胞分离，UAS-GFP飞线无SYTOX -设置此参数（步骤4.4.3），确保所有的GFP阳性细胞散点图中绘制。如果这些参数设置不正确，将分选的细胞群体将最有可能含有的死细胞和碎片（ 图5B，C）中，许多GFP阳性细胞将被错过（ 图5D）和两个不同的峰为GFP阳性细胞作为见于直方图（ 图2E和图3E）将不会被检测到。一旦FACS和门控参数设置，仪器校准并准备进行排序从ESG -GAL4，UAS-GFP飞线细胞。由于这些参数可以保存，将来所有的ESG -GAL4，UAS-排序为ISC的和EBS会议GFP飞线从4.5步开始。虽然选择了"纯度"模式和执行双向纯度排序降低细胞排序的数量，它允许一个较高排序严紧（步骤4.6和图4B，C）。值得注意的是，使用代替碘化SYTOX标记死细胞的优点是，仅存在低的溢出到FITC（绿色荧光蛋白）的信道，这使得它易于以补偿外溢。 我们的富集ISC的和胚的方法，分别是基于根据不同的GFP表达水平排序的单元格。这可能是因为老化misdifferentiated的EB也表达GFP在较低的水平，并可能被误认为ISC的期间。然而，由于分选的细胞也有不同的尺寸和粒度，我们相信，我们的排序策略是该日期最合适的富集ISC的和胚。此外，已知的是在细胞老化中肠保持ISC身份具有小核15。以获得对分选的细胞的身份更加清楚，人们可以进行排序从转基因飞线承载ESG -GAL4，UAS-GFP和Notch信号传导记者转基因的细胞。由于陷波已被证明是有效只在胚12,13，ISC的从年轻肠分离将是负的缺口记者，而胚将利好所述Notch报道。然而，在老化Notch信号变为异常的肠组织。因此，它需要被测试的这个实验设置是否确实是更可靠的ISC的和EBS区分从旧肠中分离。 我们已经采用这种方法来ISC的比较转录组分析，从年轻人和老年人肠和老化过程中发现了一些差异调节因子（unpub。observ）。此外，该方法允许用于分析ISC的与胚之间的基因表达差异。这种调查会有可能提供深入了解作为细胞进入分化过程中发生的最初的分子变化。此外，EB的过程中老化和后续分析隔离将揭示年龄诱导misdifferentiation的分子变化的光。此外，该方法可以与用于在果蝇来研究基因功能的其它遗传工具相结合。总之，这种方法提供了一个不带偏见的办法衰老过程中，调查的分子特征和ISC的和EBS的变化。这是一个有价值的工具，将促进衰老机制在成体干细胞和祖细胞的探索。

老去的一个不可避免的后果是组织和器官，以保持工作的能力下降。成年干细胞是用于维持组织稳态和器官的功能必不可少的，然而作为生物体年龄，干细胞也经历在其生物学行为的下降。这是特别有害的组织具有高周转率，如肠上皮。老化的干细胞的标志，包括基因组损伤，受损修复机制，受损的细胞周期调控和misregulated信号通路，所有这些都影响正常干细胞的行为（在1-3中综述）。通过操纵特定的信号转导通路或撞倒的特定基因，我们在规范和维护正常干细胞的行为获得洞察他们的角色。由于大多数这些实验是候选分子的方法，我们对过程中发生的在干细胞中的内源性分子变化知之甚少老化。处理这个问题的一种方法是比较年轻的转录与旧的干细胞，以确定分子的表达谱的老化过程中显著变化。不幸的是，生物和技术挑战阻碍了我们对这种做法至今进展。 果蝇是高度合适的模式生物，研究老化，因为它具有很短的寿命（约60-70天），并表现出衰老表现型（以4中综述）。此外，在果蝇的老化过程可以通过温度来加速。当保持苍蝇在29°C，在肠组织的老年型已经可以在15天后观察到5。此外， 果蝇是适合的大量遗传操作的。特别是，在果蝇中肠已成为一个很好的模型系统，研究不同的信号通路，并在环境挑战的影响肠道干细胞（ISCS）的老化过程的生物学（6-10综述）。 果蝇肠上皮具有 ​​较高的周转率，并每月更新每两周在女性和大约一次的男性11。居住在果蝇中肠ISC的具有分裂并产生一个自我更新的ISC和一个称为enteroblast（EB）12,13有丝分裂后前体细胞的能力。在EB分化为任一的吸收性肠细胞或分泌肠内分泌细胞。在此日期，唯一的标记物的组合，明确标记的ISC是转录因子田螺（ESG）和Notch配体德尔塔（DL）14的表达。然而，这仅持有如此年轻和健康的肠道。期间老化，中肠上皮的特征在于增加的ISC增殖15-17。此外，异常Notch信号扰乱的ISC子细胞的命运决定并诱导胚15 misdifferentiation。这导致细胞，激活Notch信号和共表达ESG和D1的积累，从而使去离子表达不足以识别真正的干细胞在老化中肠。在确定真正的ISC的困难阻碍了研究内生变化，老龄化ISC的到现在为止的能力。 我们已通过采取ESG -Gal4，UAS-GFP转基因的飞线，其中在ISC的和胚的GFP表达水平是本质上的不同，并保持在整个老化不同的优点解决这个问题。类似的方法已被描述为幼虫神经母细胞和神经元18,19的隔离。年轻人和老年人ESG -Gal4，UAS-GFP苍蝇肠解剖和分离成单个细胞。然后将细胞进行排序，使用荧光激活细胞分选（FACS）的GFP阳性细胞。有趣的是，排序GFP阳性ÇELLS分布到基于GFP荧光强度两个不同的峰（GFP 高和GFP 低 ）。此外，GFP阳性细胞中的两个峰也与细胞大小分布:即显示出低的GFP强度将细胞小，而细胞具有高的GFP强度较少的颗粒是较大的和更精细。该观察结果表明，较小的ISC的可能从使用基于GFP的强度的FACS和选择适当的前向散射（FSC）和侧向散射（SSC）设置较大的EB进行区分。有趣的是，两峰仍然陈酿过程中明确分开。此外，两峰的比率的方式，反映老化肠的上述标志发生变化:即大的数量，misdifferentiated的EB随时间增加。从这些研究结果，我们得出结论，通过使用适当的FACS参数设置GFP阳性细胞分选，我们可以在任何时间点d富集ISC的和胚uring老化。 综上所述，我们引入了FACS战略，使研究人员能够充实了两个不同的细胞群，ISC的和EBS，从任何年龄的果蝇肠和隔离这些细胞进行进一步的分析，如新一代测序。这种强大的方法允许研究所固有的在干细胞或祖细胞的富集人口老龄化的内源性分子机制。从这些研究中获得的数据，无疑会促进保守分子，在跨物种老化显著的鉴定。

<table><tbody><tr><td>Forceps: Dumont, Inox Biologie #5</td><td>Fine Science Tools</td><td>11252-20</td><td></td></tr><tr><td>SefarNitex 03-150um/38 (35 &amp;micro;m nylon mesh)</td><td>Sefar</td><td>3A03-0150-102-00</td><td></td></tr><tr><td>Falcon 5 ml Round Bottom Polystyrene Test Tube with Cell Strainer Snap Cap</td><td>Corning</td><td>352235</td><td></td></tr><tr><td>Polymax 1040</td><td>Heidolph</td><td>543-42210-00</td><td></td></tr><tr><td>Albumin from bovine serum (BSA)</td><td>Sigma</td><td>A4503-50G</td><td></td></tr><tr><td>0.5% Trypsin-EDTA</td><td>Invitrogen</td><td>15400-054</td><td>Trypsin obtained from a different company most likely has a different activity and the duration of the trypsin digest has to be adjusted accordingly.</td></tr><tr><td>SYTOX Blue Dead Cell Stain for flow cytometry</td><td>Life Technologies</td><td>S34857</td><td></td></tr><tr><td>RNAlater Stabilization Solution</td><td>Life Technologies</td><td>AM7023</td><td>Other solutions, &lt;em&gt;e.g.&lt;/em&gt;, Trizol can be used for subsequent RNA isolation</td></tr><tr><td>FACSAria II cell sorter</td><td>Becton Dickinson</td><td></td><td>Turn on one hour prior to sorting</td></tr></tbody></table>

isolating intestinal stem cells from adult drosophila midguts by facs to study stem cell behavior during aging

Aging tissue is characterized by a continuous decline in functional ability. Adult stem cells are crucial in maintaining tissue homeostasis particularly in tissues that have a high turnover rate such as the intestinal epithelium. However, adult stem cells are also subject to aging processes and the concomitant decline in function. The Drosophila midgut has emerged as an ideal model system to study molecular mechanisms that interfere with the intestinal stem cells&#8217; (ISCs) ability to function in tissue homeostasis. Although adult ISCs can be easily identified and isolated from midguts of young flies, it has been a major challenge to study endogenous molecular changes of ISCs during aging. This is due to the lack of a combination of molecular markers suitable to isolate ISCs from aged intestines. Here we propose a method that allows for successful dissociation of midgut tissue into living cells that can subsequently be separated into distinct populations by FACS. By using dissociated cells from the esg-Gal4, UAS-GFP fly line, in which both ISCs and the enteroblast (EB) progenitor cells express GFP, two populations of cells are distinguished based on different GFP intensities. These differences in GFP expression correlate with differences in cell size and granularity and represent enriched populations of ISCs and EBs. Intriguingly, the two GFP-positive cell populations remain distinctly separated during aging, presenting a novel technique for identifying and isolating cell populations enriched for either ISCs or EBs at any time point during aging. The further analysis, for example transcriptome analysis, of these particular cell populations at various time points during aging is now possible and this will facilitate the examination of endogenous molecular changes that occur in these cells during aging.

从FAC 50,000-100,000细胞分选，160和200肠之间获得需要进行解剖。显然，这是最耗时的整个过程中的步骤。在图1A所示的卡通图示肠道夹层，其中详细这里提供的协议描述的主要步骤。此过程可以单独修改。甲解剖，整个消化道示于图1B。 当所有的肠组织已被消化，立即继续FAC排序。继在协议中所述的门策略使得细胞群富集ISC的和胚从年轻的（ 图2）的成功识别和分类 ​​，并从旧肠（ 图3）。则P1栅极被设置为只包括健康的活细胞，以及栅极出死细胞。细胞绘制超过10 3的对数Y型斧在使用太平洋蓝色通道被定义为死细胞。该下面40上的X轴（FSC-A）的曲线图大多碎片，因此该点也被排除在外（ 图2A）。在SSC-A的散点图与FSC-A的电池是基于它们的大小和粒度分布。为获得最佳的分辨率，将细胞放置在散点图如通过调整光电倍增管（PMT）的电压示于图2B。在散点图FSC-H与FSC-W和SSC-H与SSC-W单细胞从基于尺寸（ 图2C）聚集体和双峰分离并基于粒度（ 图2D）。当描绘包括在栅极P4在直方图，GFP阴性，GFP 低 （栅极P5）的独特分离和GFP 高 （栅极P6）细胞中的细胞是可见的（ 图2E）。细胞表现出较低的GFP强度是小的和较少的颗粒，并代表ISC的。细胞表现出较高的GFP INT密度更大，更精细的和表示的EB。上的cDNA由RNA合成各GFP阳性种群细胞逆转录酶（RT）-PCR用于DL（门P5，P6）表明所述DL信号是在较小的， 低的GFP细胞比在较大的，GFP 高确实更强细胞（ 图2H）。然后将细胞backgated并在等高线图中描绘，以确认该GFP 低 （栅极P5）和GFP 高 （栅极P6）细胞不同尺寸和粒度（ 图2F）和仍然活着（ 图2G）。值得注意的是，GFP阳性细胞的两个峰（GFP 低和GFP 高 ）只能是很好分辨如果FITC（绿色荧光蛋白）的信道已经校准使用在协议中描述的控制正常。 整理旧胆量（ 图3）衍生ISC的当同一门控策略使用。散点图（ 造型玩具ê3A-D）中，直方图（ 图3E）和等高线图（ 图3F，G）的 ​​类似于图2中所示的那些。如以上所提到的，不存在真正的标记物，以确定ISC的旧肠，因此没有RT-PCR数据在这里呈现。然而，耐人寻味的观察这里是含GFP 低 （门P5）或GFP 高 （门P6）细胞的两峰老化过程中保持明显分开。此外，细胞在GFP 高 （栅极P6）峰的数目老化，这清楚地模拟misdifferentiated的EB随着年龄的已知累积期间显著增加。在这两个细胞群体的比率的变化，也可以在散点图看出（对比图3A-D和图2A-D）和在所述等高线图（比较图3F，G与图2F，G）。从这些发现，我们的结论是通过对GFP的排序使用FACS阳性细胞，我们可以在年轻人和老年人肠富集ISC的和EBS。 为了验证隔离ISC的和EBS的纯度，后排序分析进行（ 图4）。该直方图后整理ISC的和EBS（ 图4B，C）中描绘的95％和98％之间的纯度。 如果设置如上所述的FACS参数的层次是严格遵循，这两种不同的细胞群（GFP 低 ，体积小和GFP 高 ，大）已经可以在散点图的GFP与FSC-A（ 图5A）不同。这两个细胞群体无法区分，如果这个层级被忽略（ 图5B-D）。 <img alt="图1" src="/files/ftp_upload/52223/52223fig1highres.jpg" /> 图1:（A）的狄氏的主要步骤示意图电视机飞肠。首先，头部被移除（除去翼是可选的），接着在胸部和腹部，以暴露肠道的分离。肠道随后从体腔中解脱出来，在下面的步骤。黑箭头显示解剖的进展，而暗红色箭头表示拉伸方向。在最后一个图像的红线表示中肠的前部和后部的边界。（B）的成年果蝇肠道的光学显微镜图像。主要区域和解剖特征被标记。红线标记肠的边界。 <img alt="图2" src="/files/ftp_upload/52223/52223fig2highres.jpg" /> 图2:被FAC排序策略，识别和年轻的（7天）肠隔离ISC的和EBS为了更好的可视化，细胞较ISC的以红色突出显示，并代表胚细胞突出在散点图（AD）的蓝色和在等高线图（F，G）。（A）中的 P 1的栅极被设置为排除以上10 3绘制在对数Y轴上死，SYTOX阳性细胞。在FSC-A的阈值设置在40至还排除死细胞和碎片。（B）细胞的门FSC-A和SSC-A根据大小和粒度（门P2）选择单元格。该直方图（E）用于并联识别GFP阳性细胞的FSC-A对SSC-A散点图（B），（C，D）的散点图FSC-H与FSC-W和SSC-H与SSC-W用来除去团粒，双峰，并选择单峰（在C栅极P3和P4的栅极在D中）。（e）该直方图示出了细胞和它们的GFP强度的数目。两个峰值，绿色荧光蛋白低 （栅极P5）和GFP 高 （栅极P6）能够被区分。（F，G）将细胞回门控中的等高线图，以验证该两个不同的GFP阳性细胞populat离子是正确的尺寸（F）的和含有活细胞（G）。（H）的逆转录酶（RT）-PCR用于DL上的cDNA从存在于第一峰（栅极P5）的细胞中分离的RNA合成的，并在第二峰（栅极P6）分别。可在细胞出现在门与P5 P6门被检测更多去离子表达。微管蛋白担任装载控制。 <a href="https://www.jove.com/files/ftp_upload/52223/52223fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52223/52223fig3highres.jpg" /> 图3:FAC排序策略，以识别和旧的（60天）肠隔离ISC的和EBS为了更好的可视化，是代表ISC的细胞以红色突出显示，并代表胚细胞蓝色的散点图（AD）突出和。等高线图（F，G）。（A）中的 P 1的栅极被设置为排除以上10 3绘制在对数Y轴上死，SYTOX阳性细胞。在FSC-A的阈值设置在40至还排除死细胞和碎片。值得注意的是，在较大的GFP阳性细胞的年龄依赖性增加已经是明显的在此情节。（B）的细胞门控为FSC-A和SSC-A根据尺寸和粒度（栅极P2）来选择细胞。该直方图（E）用于并联识别GFP阳性细胞的FSC-A对SSC-A散点图（B），（C，D）的散点图FSC-H与FSC-W和SSC-H与SSC-W用来除去团粒，双峰，并选择单峰（在C栅极P3和P4的栅极在D中）。（e）该直方图示出了细胞和它们的GFP强度的数目。两个峰值，绿色荧光蛋白低 （栅极P5）和GFP 高 （栅极P6）能够被区分。值得注意的是，含有较大的细胞的细胞群GFP 高增加了我在老龄化n个。（F，G）细胞，背门在等高线图来验证两个不同的GFP阳性的细胞群体是正确的大小（F）和含有活细胞（G）。 <a href="https://www.jove.com/files/ftp_upload/52223/52223fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52223/52223fig4highres.jpg" /> 图4:A类后分析，以确定孤立ISC的和EBS的纯度（A）GFP阳性细胞从年轻（7天），老（60天）肠是FAC整理和直方图显示它们的分布到基于GFP强度两个峰值。（B）该职位排序分析表明孤立ISC的从年轻人和老年人肠是&gt; 97％的纯度。（C）排序电子商务的后期排序分析学士学位，年轻人和老年人肠显示&gt; 95％的纯度。细微的杂质可能是由于荧光猝灭或因重新排序的细胞机械损伤GFP表达细胞。 <a href="https://www.jove.com/files/ftp_upload/52223/52223fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52223/52223fig5highres.jpg" /> 图5:该显示不理想的FACS数据，当未设置所述的FACS参数适当地被视为散点图实施例（A）的代表性FACS所有小区的信息时的FACS参数分别设定适当的基础上在该协议中描述的对照。含ISC的细胞群体是红色圆圈和含胚细胞群体是盘旋在蓝色。盘旋在绿色的细胞是死细胞具有更强的自体荧光相比，活细胞，其被绘制低于10 3上的对数y轴。（B）散点图显示了当的FACS仪器未校准获得的典型结果。没有检测到GFP阳性细胞。（℃）如果FSC设置不正确，未检测到不同的GFP阳性细胞群。（D）中如果FITC（绿色荧光蛋白）信道不正确调整，大部分GFP-的进行排序阳性细胞散点图，而是沿着剧情的上边缘不会绘制。另外，在此处示出的曲线图，该限制为自发荧光不调节，因此，细胞积高于10 2上的对数y轴实际上是自体荧光和可能被误认为是GFP阳性细胞。

Watch this Scientific Journal Video about Isolating Intestinal Stem Cells from Adult Drosophila Midguts by FACS to Study Stem Cell Behavior During Aging at JoVE.com

Isolating Intestinal Stem Cells from Adult Drosophila Midguts by FACS to Study Stem Cell Behavior During Aging

Understanding the endogenous molecular changes in adult stem cells during aging requires isolating the cells of interest. The method described here presents a simple and robust approach to enrich for and isolate Drosophila intestinal stem cells and the enteroblast progenitor cells by FACS at any time point during aging.

隔离成人肠道干细胞<em&gt;果蝇</em&gt;肠用流式细胞仪研究干细胞的行为在老化

Aging tissue is characterized by a continuous decline in functional ability. Adult stem cells are crucial in maintaining tissue homeostasis particularly ...

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Research

JoVE Journal

Biology

Isolating Intestinal Stem Cells from Adult Drosophila Midguts by FACS to Study Stem Cell Behavior During Aging

20.1K 次观看.  Universität Ulm. Understanding the endogenous molecular changes in adult stem cells during aging requires isolating the cells of interest. The method described here presents a simple and robust approach to enrich for and isolate Drosophila intestinal stem cells and the enteroblast progenitor cells by FACS at any time point during aging. 

视频: Isolating Intestinal Stem Cells from Adult Drosophila Midguts by FACS to Study Stem Cell Behavior During Aging

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

该<em&gt;果蝇</em&gt;成虫盘瘤模型:基因的表达与肿瘤侵袭性的可视化和定量遗传马赛克

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

科研

癌症研究

A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands

To understand the function of an organ, it is often useful to understand the role of its constituent cell populations. Unfortunately, the rarity of individual cell populations often makes it difficult to obtain enough material for molecular studies. For example, the accessory gland of the Drosophila male reproductive system contains two distinct secretory cell types. The main cells make up 96% of the secretory cells of the gland, while the secondary cells (SC) make up the remaining 4% of cells (about 80 cells per male). Although both cell types produce important components of the seminal fluid, only a few genes are known to be specific to the SCs. The rarity of SCs has, thus far, hindered transcriptomic analysis study of this important cell type. Here, a method is presented that allows for the purification of SCs for RNA extraction and sequencing. The protocol consists in first dissecting glands from flies expressing a SC-specific GFP reporter and then subjecting these glands to protease digestion and mechanical dissociation to obtain individual cells. Following these steps, individual, living, GFP-marked cells are sorted using a fluorescent activated cell sorter (FACS) for RNA purification. This procedure yields SC-specific RNAs from ~40 males per condition for downstream RT-qPCR and/or RNA sequencing in the course of one day. The rapidity and simplicity of the procedure allows for the transcriptomes of many different flies, from different genotypes or environmental conditions, to be determined in a short period of time.

A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands

Here, we present a protocol to dissociate and sort a specific cell population from the Drosophila male accessory glands (secondary cells) for RNA sequencing and RT-qPCR. Cell isolation is accomplished through FACS purification of GFP-expressing secondary cells after a multistep-dissociation process requiring dissection, proteases digestion and mechanical dispersion.

从果蝇雄性附件腺的二级细胞中分离RNA的FACS协议

To understand the function of an organ, it is often useful to understand the role of its constituent cell populations. Unfortunately, the rarity of ...

遗传学

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and differentiation. Typically, pHi dynamics are determined in cultured cells, which are amenable to measuring and experimentally manipulating pHi. However, the recent development of new tools and methodologies has made it possible to study pHi dynamics within intact, live tissue. For Drosophila research, one important development was the generation of a transgenic line carrying a pHi biosensor, mCherry::pHluorin. Here, we describe a protocol that we routinely use for imaging live Drosophila ovarioles to measure pHi in the epithelial follicle stem cell (FSC) lineage in mCherry::pHluorin transgenic wild type lines; however, the methods described here can be easily adapted for other tissues, including the wing discs and eye epithelium. We describe techniques for expressing mCherry::pHluorin in the FSC lineage, maintaining ovarian tissue during live imaging, and acquiring and analyzing images to obtain pHi values.

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

We provide a protocol for imaging intracellular pH of an epithelial stem cell lineage in live Drosophila ovarian tissue. We describe methods to generate transgenic flies expressing a pH biosensor, mCherry::pHluorin, image the biosensor using quantitative fluorescence imaging, generate standard curves, and convert fluorescence intensity values to pH values.

活体果蝇卵巢组织中卵泡干细胞谱系的细胞内 pH 值的成像方法

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and ...

发育生物学

Dissection and Staining of Drosophila Pupal Ovaries

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a pupal case. The challenge of dissecting pupal ovaries also lies in their physical location within the pupa: the ovaries are surrounded by fat body cells inside the pupal abdomen, and these fat cells must be removed to allow for proper antibody staining. To overcome these challenges, this protocol utilizes customized Pasteur pipets to extract fat body cells from the pupal abdomen. Moreover, a chambered coverglass is used in place of a microcentrifuge tube during the staining process to improve visibility of the pupae. However, despite these and other advantages of the tools used in this protocol, successful execution of these techniques may still involve several days of practice due to the small size of pupal ovaries. The techniques outlined in this protocol could be applied to time course experiments in which ovaries are analyzed at various stages of pupal development.

Dissection and Staining of Drosophila Pupal Ovaries

The Drosophila ovary is an excellent model system for studying stem cell niche development. Though methods for dissecting larval and adult ovaries have been published, pupal ovary dissections require different techniques that have not been published in detail. Here we outline a protocol for dissecting, staining, and mounting pupal ovaries.

果蝇蛹卵巢的解剖与染色

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, germ cell development, piRNA biology, and niche formation. Here, we present a dissection technique to live-image the gonad ex vivo during a period when in vivo live-imaging is highly ineffective. This protocol outlines how to transfer embryos to an imaging dish, choose appropriately-staged male embryos, and dissect the gonad from its surrounding tissue while still maintaining its structural integrity. Following dissection, gonads can be imaged using a confocal microscope to visualize dynamic cellular processes. The dissection procedure requires precise timing and dexterity, but we provide insight on how to prevent common mistakes and how to overcome these challenges. To our knowledge this is the first dissection protocol for the Drosophila embryonic gonad, and will permit live-imaging during an otherwise inaccessible window of time. This technique can be combined with pharmacological or cell-type specific transgenic manipulations to study any dynamic processes occurring within or between the&#160;cells in their natural gonadal environment.

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

Here, we provide a dissection protocol required to live-image the late embryonic Drosophila male gonad. This protocol will permit observation of dynamic cellular processes under normal conditions or after transgenic or pharmacological manipulation.

晚期胚胎 果蝇 性腺的解剖和活体成像

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

夹层和染色果蝇幼虫卵巢

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

生物学

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

分离果蝇睾丸

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Real-Time Imaging of Germline Stem Cells in Drosophila Ex Vivo Germaria Culture

Extended Live Imaging of Female Drosophila melanogaster Germline Stem Cell Niches

Live imaging methods allow the analysis of dynamic cellular processes in detail and in real-time. The Drosophila ovary represents an excellent model to explore the dynamics of a myriad of developmental processes, such as cell division, stemness, differentiation, migration, apoptosis, autophagy, cellular adhesion, etc., over time. Recently, we have implemented an extended ex vivo culture and live imaging of the female Drosophila GSC niche. Using a Drosophila line harboring a GFP::Par-1 transgene as an example, this method allows the visualization of the GSCs&#39; asymmetric division within their niche and the description of the changes in the spectrosome morphology along the cell cycle. Here, we present a detailed protocol for the ex vivo culture of Drosophila germaria, enabling prolonged visualization of the female GSC niche. Importantly, this protocol is broadly applicable to live imaging GSCs with multiple fluorescently tagged proteins of interest that are available in stock centers and/or in the Drosophila research community.

Here, we provide a detailed protocol to perform live imaging of the asymmetric division of germline stem cells (GSCs) in the Drosophila ovarian niche. We use a transgenic line that ubiquitously expresses a green fluorescent protein (GFP) fusion of the spectrosome protein Par-1.

Live imaging methods allow the analysis of dynamic cellular processes in detail and in real-time. The Drosophila ovary represents an excellent model to ...

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin&#8212;the histone-to-protamine switch.
Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages.
The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre- and post-meiotic spermatogenesis.

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

This protocol describes the dissection and cultivation of intact testes and germ-line cysts from Drosophila melanogaster pupae. This method allows microscopic observation of spermatogenesis ex vivo. Furthermore, we describe a pharmacological assay of the effect of inhibitors on specific stages of germ-cell development in pupal testes.

解剖，成像和药物治疗: 果蝇蛹睾丸和单身男性生殖系囊肿的体外培养

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes ...

Stimulating and Analyzing Adult Neurogenesis in the Drosophila Central Brain

The molecular and cellular mechanisms underlying neurogenesis in response to disease or injury are not well understood. However, understanding these mechanisms is crucial for developing neural regenerative therapies. Drosophila melanogaster is a leading model for studies of neural development but historically has not been exploited to investigate adult brain regeneration. This is primarily because the adult brain exhibits very low mitotic activity. Nonetheless, penetrating traumatic brain injury (PTBI) to the adult Drosophila central brain triggers the generation of new neurons and new glia. The powerful genetic tools available in Drosophila combined with the simple but rigorous injury protocol described here now make adult Drosophila brain a robust model for neural regeneration research. Provided here are detailed instructions for (1) penetrating injuries to the adult central brain and (2) dissection, immunohistochemistry, and imaging post-injury. These protocols yield highly reproducible results and will facilitate additional studies to dissect mechanisms underlying neural regeneration.

Stimulating and Analyzing Adult Neurogenesis in the Drosophila Central Brain

This article provides detailed protocols for inflicting Penetrating Traumatic Brain Injury (PTBI) to adult Drosophila and examining the resulting neurogenesis.

刺激和分析 果蝇 中枢脑中的成人神经发生

The molecular and cellular mechanisms underlying neurogenesis in response to disease or injury are not well understood. However, understanding these ...

隔离成人肠道干细胞

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从果蝇雄性附件腺的二级细胞中分离RNA的FACS协议

活体果蝇卵巢组织中卵泡干细胞谱系的细胞内 pH 值的成像方法

果蝇蛹卵巢的解剖与染色

晚期胚胎果蝇性腺的解剖和活体成像

夹层和染色果蝇幼虫卵巢

分离果蝇睾丸

Extended Live Imaging of Female Drosophila melanogaster Germline Stem Cell Niches

解剖，成像和药物治疗: 果蝇蛹睾丸和单身男性生殖系囊肿的体外培养

刺激和分析果蝇中枢脑中的成人神经发生

隔离成人肠道干细胞

摘要

探索更多视频

此视频中的章节

该

从果蝇雄性附件腺的二级细胞中分离RNA的FACS协议

活体果蝇卵巢组织中卵泡干细胞谱系的细胞内 pH 值的成像方法

果蝇蛹卵巢的解剖与染色

晚期胚胎 果蝇 性腺的解剖和活体成像

夹层和染色果蝇幼虫卵巢

分离果蝇睾丸

Extended Live Imaging of Female Drosophila melanogaster Germline Stem Cell Niches

解剖，成像和药物治疗: 果蝇蛹睾丸和单身男性生殖系囊肿的体外培养

刺激和分析 果蝇 中枢脑中的成人神经发生

晚期胚胎果蝇性腺的解剖和活体成像

刺激和分析果蝇中枢脑中的成人神经发生