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。

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			<td height="42" style="height:42px;width:271px;">Forceps: Dumont, Inox Biologie #5</td>
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			<td style="width:105px;">Invitrogen</td>
			<td style="width:104px;">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>
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			<td height="42" style="height:42px;width:271px;">SYTOX Blue Dead Cell Stain for flow cytometry</td>
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			<td style="width:104px;">S34857</td>
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			<td height="42" style="height:42px;width:271px;">RNAlater Stabilization Solution</td>
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			<td style="width:104px;">AM7023</td>
			<td>&#160;other solutions, e.g. Trizol can be used for subsequent RNA isolation</td>
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			<td height="42" style="height:42px;width:271px;">FACSAria II cell sorter</td>
			<td style="width:105px;">Becton Dickinson</td>
			<td style="width:104px;"></td>
			<td>Turn on one hour prior to sorting</td>
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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 ...

isolating-intestinal-stem-cells-from-adult-drosophila-midguts-facs-to

Research

JoVE Journal

Biology

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

19.9K 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

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

在成年斑马鱼的全肾骨髓移植

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

科研

生物学

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

从小鼠胚胎干细胞的造血干细胞的推导

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

人类胚胎干细胞的推导Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

成人内皮细胞集落形成祖细胞的分离和大型规模扩张

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolating Stem Cells from Soft Musculoskeletal Tissues

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of excitement for treating injured and diseased tissues due to their impressive capabilities to undergo multi-lineage cell differentiation and their self-renewal ability. Most importantly, these qualities have made them advantageous for use in autologous cell transplantation therapies. The current protocol will introduce the readers to the modified preplate technique where soft tissues of the musculoskeletal system, e.g. tendon and muscle, are 1st enzymatically dissociated and then placed in collagen coated flasks with medium. The supernatant, which is composed of medium and the remaining floating cells, is serially transferred daily to new flasks. The stem cells are the slowest to adhere to the flasks which is usually takes 5-7 days (serial transfers or preplates) . By using this technique, adult stem cells present in these tissues can be easily harvested through fairly non-invasive procedures.

Isolating adult stem cells from musculoskeletal soft tissues based on the cell's adherence speed to flask.

从软肌肉骨骼组织中分离干细胞

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

人类胚胎干细胞畸胎瘤形成命运的映射

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

选择和隔离殖民地的人类诱导多能干细胞重新编程成年成纤维细胞

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

单个肌纤维和成人骨骼肌卫星细胞的分离和培养

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

天真成人干细胞的分离从初级人类成纤维细胞培养

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...