笔者想感谢细胞分离方法和凯文·韦勒，大卫·弗莱厄蒂和布列塔尼马特洛克建议凯瑟琳·奥尔福德在范德比尔特大学医学中心和Melissa A.马瑟在流式细胞仪共享资源的支持与插图艺术的援助。我们感谢博士。杰克·毛思迪和肖恩·莫里森实施隔离的神经祖细胞的意见。 VMC流式细胞仪共享资源是由范德比尔特英格拉姆癌症中心（P29 CA68485）和范德比尔特消化系统疾病研究中心（P29 DK058404）的支持。这项工作是由来自美国国家卫生补助DK064251，DK086594，DK070219研究院的资金支持。

1。媒体的准备（所有步骤在做组织培养罩） <ol><li>结合以下几点：44毫升的L-15中，0.5毫升100X青霉素/链霉素（的P / S），0.5毫升100毫克/毫升牛血清白蛋白（BSA），0.5毫升1M肝素钠，5毫升的组织培养级水。务必混合，然后加入BSA和​​P / S彻底。这个量应该是足够了分解到5个不同类型的组织。该媒体将使用在步骤3.4准备淬火组织酶解后使用的解决方案。 </li><li>虽然0.22微米聚醚砜（PES）过滤器的过滤介质。 </li><li>准备1X汉克的平衡盐溶液（HBSS中）和1X的磷酸盐缓冲液（PBS）10X股票，利用组织培养级水。通过0.22微米的PES过滤器过滤。大量的这些试剂可以提前准备的时间，并储存在4°C，aliquoting需要在组织文化引擎盖小卷。 </li><li>填写1X的HBSS（多个15毫升锥形管数等于你打算分，解剖组织）保持冰管的管。 </li></ol> 2。解剖<ol><li>按照机构的动物护理和使用委员会批准的协议和传输子宫安乐死定时到60或100毫米培养皿中含有冰冷的1×PBS孕鼠。 </li><li>从子宫中取出胚胎及安乐死在冰冷的1×PBS斩首。屏幕荧光照明下单独划分为阳性转基因和野生型（WT）的非转基因池。整个解剖胚胎保存在冰冷的1×PBS。 </li><li>在解剖显微镜下，分解剖泌尿生殖道。举行的地方用细镊子前肢的胚胎。从肝脏中取出内脏，生殖器结节水平膜片钳插入，然后轻快地拉动，远离背体壁内脏器官。 </li><li>进一步细分，解剖组织远离利益的周围组织（ 图1）。放入15毫升锥形管含冰冷的1X HBSS中的每个分解剖组织类型。池每个组织类型根据胚胎型（ 即所有GFP +胎儿小肠样本集中在一个单一的15毫升锥形管）。 </li><li>同时，从野生型胚胎解剖相媲美的组织补偿控制使用流式细胞仪。 </li></ol> 3。解离Subdissected组织<ol><li>颗粒子的解剖组织210相对离心力（RCF），4℃离心5分钟。离心后，吸出尽可能多的HBSS。 </li><li> Accumax（创新的细胞技术公司）悬浮颗粒组织，一定要改变每个样品之​​间的枪头，组织类型，以避免交叉污染。 Accumax增值金额为数额较大或较小的组织可以扩大。通常为1将用于-5胎儿小肠样本，1毫升Accumax，但更大的组织池将需要更大的体积来实现分离。 </li><li>地方管到37°C的水浴20-45分钟，取决于被孤立的组织（ 如 13.5 DPC肠20分钟，15.5 DPC肠道35分钟）的阶段。中途虽然分离时间，组织对水浴（或任何固体表面）一边敲管和“弹”管（会动摇了一个老式的水银温度计）手动分手。在解离时间结束，下跌过程中多次重复的震动，进一步分解样品。对于较脆弱的样品，减少摇下来的蓬勃生机，而不是在步骤3.5中使用吸管研磨，以达到适当的分解水平。典型的解离为14.5 DPC和15.5 DPC LUT的时间是35和45分钟分别。 </li><li>虽然组织孵化Accumax，淬火，使lutions，称为淬火和淬火1:5。淬火时，加入45μL5 mg / ml的DNA酶I至6毫升的L-15媒体。加入45μL5 mg / ml的DNA酶I至30毫升的L-15媒体解渴1:5。 </li><li>在解离结束时，走上冰15 ml反应管，并立即加入1毫升的淬火，以每管。每个样品磨碎，直到组织几乎完全分离（ 图2）。仍然会有组织在解决目前一些小块。实现一个完全同质的样本，是既不容易实现的，也不可取，因为它可以导致细胞活力差。 </li><li>冰样品，尽可能保持协议的其余部分。时移液淬火或淬火1:5到避免样品的交叉污染，务必使用新鲜枪头。 </li></ol> 4。过滤细胞悬液<ol><li>使用已在70％乙醇浸泡的镊子，将在38微米尼龙网膜3厘米正方形（SEFAR美国）一个新的15毫升锥形管口。通过滤网吸取到中心使用窄口径的提示膜细胞。如果趋于饱和，而过滤膜，将它删除，干管口与kimwipe，并使用一条新的尼龙网过滤细胞悬液的其余部分。一旦所有的细胞已被过滤，用1毫升，淬火1:5到冲洗管和过滤任何剩余的细胞。 </li><li>过滤时是完整的，或更换堵塞膜时，使用镊子卷起网格的两侧和整个管顶部擦拭膜细胞中删除的任何挂水滴。 </li><li>在颗粒细胞悬液，离心，4℃5分钟210rcf。 1毫升淬火1:5吸上清，重悬沉淀。 </li><li>通过尼龙网过滤细胞悬液分为5毫升聚苯乙烯管。洗出1毫升15毫升管淬火1:5和过滤器捕捉任何剩余的细胞。 </li></ol> 5。流式细胞仪样品准备<ol&gt; <li>如果使用排序和补偿控制相同的组织，1/10的样本量的1/20 日到一个新的5毫升聚苯乙烯管，用于补偿控制转移。野生型的组织样本分成两部分。一个部分将不染野生型（野生型），而下半年将有7-Aminoactinomycin的D类（7-AAD的，荧光intercalator的“活力染色”用活细胞除外）（7-AAD的）。这些单独的控制需要，以便赔偿之间的离散流分拣荧光发射光谱重叠。例如，对于这样一个单一的EGFP荧光记者，必要的控制措施将包括：EGFP，不染野生型细胞，并与7-AAD的（图3）染色管。 </li><li>填补所有5毫升管含有细胞悬液，以1:5和离心机淬火210rcf，4°C间5分钟。吸出上清液，留下约200微升液体在管。 </li><li> 7-AAD的1:1000稀释1:5淬火。加入7-AAD的染色稀释到7-AAD的唯一补偿控制和样本进行排序。不添加7-AAD EGFP的唯一或野生的唯一补偿控制。卷7-AAD要添加会有所不同起始原料和细胞颗粒大小获得金额为基础。一旦7-AAD的已被添加到相应的管，样品准备进行排序。 <table border="1"><tbody><tr><td> 组织 </td><td> 样品池大小 </td><td> 第7卷-AAD被添加到管* </td></tr><tr><td> 15.5 DPC小肠</td><td> 1-5 </td><td> 200μL </td></tr><tr><td> 15.5 DPC的LUT </td><td> 1-5 </td><td> 150μL </td></tr></tbody></table> 表1。自媒体的愿望是，不同的组织中分离*最后在每个管的总金额的7-AAD的添加量会有所不同数额范围从50-100微升1n个近似的过程和执行，以避免沉淀。最终卷不测量，以尽量减少处理溶液中的细胞。 </li><li>准备收集管捕获RNA提取的细胞，加入1.5毫升离心管0.75毫升的TRIzol-LS。 </li><li>如果细胞在体外培养，而不是RNA分离收集，排序，直接进入自我更新的媒体在6孔组织培养板细胞，涂有纤维连接蛋白与媒体充满如前所述。2,6,7 </li></ol> 6。流式细胞仪<ol><li>在流式细胞仪检测，评估补偿控制第一，确保每个样品之​​间冲洗，以避免污染。使用补偿样本的配置文件设置电压/门排序。请注意所需的组织细胞，如果是限制多，一个独特的组织可以用来建立补偿设置，只要长的荧光强度和细胞大小样本之间的相媲美的。 </li><li>设置补偿参数和大门，以避免占用7-AAD的染料和收集参展GFP荧光的相对强度高，不染控制（ 图4）细胞的死亡细胞。 </li><li>排序到每个离心管最大25000细胞。排序应在最低可能的压力与大口径喷嘴和低流量（ 例如 ，17psi，100微米的喷嘴，3000事件/秒），以维持神经祖细胞的可行性。为各种分离绿色荧光蛋白+细胞，我们执行我们的BD Biosciences公司FACSAriaII隔离使用20mW的488nm激光激发绿色荧光蛋白记者。 </li><li>如果样品含有很高的细胞浓度，这是明智的稀释细胞悬液，进一步用1:1000 7-AAD的染色排序时，以实现更高的捕获效率。 </li><li>涡每管细胞中的TRIzol LS排序后，立即抓获。 </li></ol> 7。代表结果组织分解产生一个细胞悬液流排序是足够的消化酶和避免过度消化，可导致细胞存活率低之间的微妙平衡。所需的组织分离的一个例子如图2所示。前手工研磨件次解剖器官组织在适当的消化仍明显（ 图2B，2F）。在太长的一段时间或酶浓度过高，是酶处理的组织，由此产生的暂停没有任何组织残留的大块（ 图2D，2H）。 手册的适当分离和过滤产生排序型材，流式细胞仪，通常表现出活菌大于90％，显示EGFP的表达水平高（ 图4）。通过这种方法得到的细胞群说明好由门捕获的EGFP +神经祖细胞的活力和可以捕获随后的文化基因表达分析。 <img alt="图1" src="/files/ftp_upload/4188/4188fig1.jpg" /> 图1。胎鼠LUT的TH-EGFP的+神经祖细胞的分布。整个安装在泌尿生殖道15.5 DPC看腹侧（一）亮场照明下EGFP的TH-EGFP的荧光照明下（二）确定的转基因表达的标记细胞的分布相比。 TH-EGFP的表达，是存在于肾上腺（a）和内侧位于腹腔神经节（CG）。横向（三）鉴于15.5 DPC TH-EGFP的亚解剖膀胱展品在盆腔神经节（PG），膀胱壁和尿道（U）（BLA）的转基因表达的荧光。背视图（D）绿色荧光蛋白+细胞是显而易见的前背尿道。其他标签：肾脏（K），睾丸（T）（BLA），膀胱和生殖结节（GT）。 <imgALT =“图2”的src =“/ files/ftp_upload/4188/4188fig2.jpg”/&gt; 图2 15 DPC胎儿的LUT（a）和小肠（E）明场图像，分别拍摄中途通过分离潜伏期，在分离培养结束前中断（B，F）手动中断后，（C， g）和样品中，已经过于分离（D，H）。 <img alt="图3" src="/files/ftp_upload/4188/4188fig3.jpg" /> 图3。示意图说明补偿控制，需要建立严格的流式细胞仪门参数。 <img alt="图4" src="/files/ftp_upload/4188/4188fig4.jpg" /> 图4。14.5 DPC（a）和15.5 DPC（二）流量排序型材的形象代表。黑人人口由单一的基础上向前和侧面分散的细胞都死了，和标记7-AAD的荧光。灰色的人口是根据已排除7-AAD的，因此可行的正向和侧面散射的单细胞组成。绿门人口表示盒装“的GFP +”区域，并已排除7-AAD的（可行的），并表现出EGFP荧光的单细胞组成。

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没有利益冲突的声明。

老鼠记者线条表达荧光记者正在成为广泛使用，通过多种努力在小鼠遗传学社区1,8,9。因此，说明这里的分离方法，可广泛应用于神经递质或受体的表达方式，无论从胎儿或成人组织为基础的离散神经元亚型隔离。虽然我们已经优化了基于荧光的转基因记者表达方法，它也可以被应用于标记活细胞免疫标记方法3-5,10表达细胞表面受体的样品。 在我们的手中，?...

The authors middle initials were omitted from the publication of <a href="http://www.jove.com/video/4188">An Optimized Procedure for Fluorescence-activated Cell Sorting (FACS) Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice</a>.

Erratum: An Optimized Procedure for Fluorescence-activated Cell Sorting (FACS) Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 供应商 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> accumax </td><td>西格玛（MFR：创新的电池技术） </td><td> A7089-100ML </td><td>存储在1毫升等分冻结</td></tr><tr><td> DNA酶I </td><td>西格玛</td><td> D - 4527 </td><td>储存在-20℃5毫克/毫升在1xHBSS， （用于在淬火，淬火1:5） </td></tr><tr><td> 10X PBS pH值7.4 </td><td> Gibco公司</td><td> 70011-044 </td><td>弥补组织文化品位水1倍，然后无菌过滤</td></tr><tr><td> 10X的HBSS W / O型钙或镁</td><td> Gibco公司</td><td> 14185-052 </td><td>弥补组织文化品位水1倍，然后无菌过滤</td></tr><tr><td>莱博维茨的L-15培养基</td><td> Gibco公司</td><td> 21083027 </td><td></td></tr><tr><td>青霉素/链霉素100X </td><td> Gibco公司</td><td>15140-133 </td><td>店分装在-20°C </td></tr><tr><td>牛血清白蛋白</td><td>西格玛</td><td> A3912-100G </td><td>储存在-20℃，100毫克/毫升的水，分装</td></tr><tr><td> biowhittaker 1M HEPES在0.85％的NaCl </td><td>龙沙</td><td> 17-737E </td><td></td></tr><tr><td> 38的微米NITEX尼龙网状膜</td><td> SEFAR美国</td><td> 3-38/22 </td><td>切成3厘米正方形。紫外线治疗一夜之间在组织文化引擎盖消毒。 </td></tr><tr><td> 7-AAD的</td><td> Invitrogen公司</td><td> A1310 </td><td> 1毫克/毫升</td></tr><tr><td> Trizol法的LS </td><td> Invitrogen公司</td><td> 10296-028 </td><td></td></tr><tr><td> 5毫升聚苯乙烯管</td><td>鹘</td><td> 352058 </td><td></td></tr><tr><td> 15毫升锥形管</td><td>康宁</td><td> 430790 </td><td></td></tr><tr><td>精解剖钳</td><td>精密科学仪器</td><td> 11251-30 </td><td>杜蒙＃5楼orcep，Dumoxel，小费标准0.1x0.06mm </td></tr><tr><td>解剖勺</td><td>精密科学仪器</td><td> 10370-18 </td><td></td></tr></tbody></table>

an optimized procedure for fluorescence-activated cell sorting (facs) isolation of autonomic neural progenitors from visceral organs of fetal mice

在开发过程中，神经嵴（数控）的神经祖细胞迁移的神经管形成自主神经节，内脏器官，如小肠和下尿路。在开发过程中，这些细胞在成熟组织往往分散的整个组织，因此，使用激光捕获微解剖的方法离散人群的隔离是很难的。不过，他们可以直接可视化，如酪氨酸羟化酶（TH）神经元特异性基因的调控区域驱动的荧光记者表达。我们描述了产量高，可行的TH +神经祖细胞从胎鼠内脏组织，包括肠道和泌尿生殖道（LUT），基于分离和荧光激活细胞分选（FACS）进行了优化的方法。 TH基因编码的限速酶生产儿茶酚胺。肠道神经祖细胞开始表达TH期间ING在胎儿小肠1和TH他们迁移也是目前在成人骨盆神经节神经元2-4的一个子集。首次亮相的血统和这些神经元在其他方面的LUT，其隔离的分布尚未描述。神经祖细胞表达TH，可以很容易地可视化表达绿色荧光蛋白在小鼠携带的基因结构的Tg（TH-EGFP）DJ76Gsat/Mmnc 1。我们在小鼠胚胎成像这个基因的表达，以文件的发展中国家的LUT 15.5天后性交（DPC）的TH的分布+细胞，划定为0.5 DPC上午插件检测，并观察到的凝聚神经祖细胞的一个子集盆腔神经节表达EGFP的。 到隔离LUT的TH +神经元祖细胞，我们优化的方法，最初用来净化神经嵴干细胞从胎鼠小肠2-6。在此之前的努力隔离NC-派生的人口依靠鸡尾酒用胶原酶和胰蛋白酶消化获得细胞悬液，流式细胞仪。在我们的手中，这些方法产生的细胞悬液，从相对较低的可行性LUT。由于胎儿的LUT组织的神经祖细胞的发病率已经很低，我们成立了优化分离方法等，将增加在最终分解细胞生存。我们决心在Accumax温柔分离（创新的细胞技术公司），手动过滤，流量在低压力，使我们能够实现一贯更大的生存（细胞总数的70％），随后产量足够的神经祖细胞下游分析排序。我们所描述的方法，可以广泛应用于多种神经元的人口，无论从胎儿或成年小鼠组织隔离。

Watch this Scientific Journal Video about An Optimized Procedure for Fluorescence-activated Cell Sorting FACS Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice at JoVE.com

An Optimized Procedure for Fluorescence-activated Cell Sorting FACS Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice

一个优化的过程，净化神经嵴源性神经祖细胞从小鼠胚胎组织被描述。此方法采用荧光报告基因的表达，通过荧光激活细胞分选（FACS）分离的离散人口。该技术可以应用到隔离整个发展或从成人组织中的神经元亚群。

荧光激活细胞分选（FACS）自主神经祖细胞的分离，从胎鼠的内脏器官的优化程序

During development neural crest (NC)-derived neuronal progenitors migrate away from the neural tube to form autonomic ganglia in visceral organs like the ...

an-optimized-procedure-for-fluorescence-activated-cell-sorting-facs

Research

JoVE Journal

Neuroscience

An Optimized Procedure for Fluorescence-activated Cell Sorting (FACS) Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice

13.6K 次观看.  Vanderbilt University School of Medicine. 一个优化的过程，净化神经嵴源性神经祖细胞从小鼠胚胎组织被描述。此方法采用荧光报告基因的表达，通过荧光激活细胞分选（FACS）分离的离散人口。该技术可以应用到隔离整个发展或从成人组织中的神经元亚群。

视频: An Optimized Procedure for Fluorescence-activated Cell Sorting FACS Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

从废弃的人类胎儿大脑皮质组织神经干细胞的生成

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

科研

神经科学

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

从小鼠胚胎神经管的神经嵴细胞的分离和培养

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.

We present an ex vivo cell migration assay that allows precise quantification of enteric neural crest cell migration potential in the presence of various growth factors.

定量细胞迁移检测方法的鼠肠神经前体细胞

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

一个鼠标，两种文化：隔离和成人神经的培养干细胞从单个小鼠的两个神经源性区

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

在大鼠脑组织用荧光活化细胞分选，以检查细胞类型特异性基因表达

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

神经干细胞和祖细胞来自成年小鼠脑室下区细胞分选和实时成像的细胞周期动力学

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific sets of sparsely distributed activated neurons called neuronal ensembles that mediate learned associations. Fluorescence Activated Cell Sorting (FACS) has recently been optimized for adult rat brain tissue and allowed isolation of activated neurons using antibodies against the neuronal marker NeuN and Fos protein, a marker of strongly activated neurons. Until now, Fos-expressing neurons and other cell types were isolated from fresh tissue, which entailed long processing days and allowed very limited numbers of brain samples to be assessed after lengthy and complex behavioral procedures. Here we found that yields of Fos-expressing neurons and Fos mRNA from dorsal striatum were similar between freshly dissected tissue and tissue frozen at -80 &#186;C for 3 - 21 days. In addition, we confirmed the phenotype of the NeuN-positive and NeuN-negative sorted cells by assessing gene expression of neuronal (NeuN), astrocytic (GFAP), oligodendrocytic (Oligo2) and microgial (Iba1) markers, which indicates that frozen tissue can also be used for FACS isolation of glial cell types. Overall, it is possible to collect, dissect and freeze brain tissue for multiple FACS sessions. This maximizes the amount of data obtained from valuable animal subjects that have often undergone long and complex behavioral procedures.

Fluorescence Activated Cell Sorting FACS and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

Here we present a Fluorescence Activated Cell Sorting (FACS) protocol to study molecular alterations in Fos-expressing neuronal ensembles from both fresh and frozen brain tissue. The use of frozen tissue allows FACS isolation of many brain areas over multiple sessions to maximize the use of valuable animal subjects.

荧光活化细胞分选（FACS）和新鲜和冷冻大鼠脑组织的Fos表达神经元基因表达分析

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific ...

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. Additionally, epigenetic chromatin modifications, like histone methylation, have an important role for establishing and maintaining specific cell fates within this process. The vast majority of histone methylation occurs on the flexible histone tail, which is accessible to histone modifiers, erasers, and histone reader proteins. In contrast, H3K79 methylation is located in the globular domain of histone 3 and is implicated in different developmental functions. H3K79 methylation is evolutionarily conserved and can be found in a wide range of species from Homo sapiens to Saccharomyces cerevisiae. The modification occurs in different cell populations within organisms, including neural progenitors. The location of H3K79 methylation in the globular domain of histone 3 makes it difficult to assess. Here, we present methods to isolate and culture cortical progenitor cells (CPCs) from embryonic cortical brain tissue (E11.5-E14.5) or cerebellar granular neuron progenitors (CGNPs) from postnatal tissue (P5-P7), and to efficiently immunoprecipitate H3K79me2 for quantitative PCR (qPCR) and genome-wide sequencing.

We present an effective and reproducible method to isolate and culture neural progenitor cells from embryonic and postnatal brain tissue for chromatin immunoprecipitation (ChIP) of histone 3 lysine 79 dimethylation (H3K79me2) - a histone mark located within the globular domain of histone 3.

组蛋白3赖氨酸 79 Dimethylation 标记沉淀后神经祖细胞的分离培养

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. ...

Primary Cell Culture of Purified GABAergic or Glutamatergic Neurons Established through Fluorescence-activated Cell Sorting

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can be cultured in defined media for 16 days in vitro and are amenable to any analyses typically performed on dissociated cultures, including electrophysiological, morphological and survival analyses. The major advantage of these cultures is that specific cell types can be selectively studied in the absence of complex external influences, such as those arising from glial cells or other neuron types. When planning experiments with purified cells, however, it is important to note that neurons strongly depend on glia-conditioned media for their growth and survival. In addition, glutamatergic neurons further depend on glia-secreted factors for the establishment of synaptic transmission. We therefore also describe a method for co-culturing neurons and glial cells in a non-contact arrangement. Using these methods, we have identified major differences between the development of GABAergic and glutamatergic neuronal networks. Thus, studying cultures of purified neurons has great potential for furthering our understanding of how the nervous system develops and functions. Moreover, purified cultures may be useful for investigating the direct action of pharmacological agents, growth factors or for exploring the consequences of genetic manipulations on specific cell types. As more and more transgenic animals become available, labeling additional specific cell types of interest, we expect that the protocols described here will grow in their applicability and potential.

This protocol describes a cell sorting based method for the purification and culture of fluorescent GABAergic or glutamatergic neurons from the neocortex and hippocampus of postnatal mice or rats.

荧光活化细胞分选建立纯化的 Gabaep 或谷胱甘肽神经元的原代细胞培养

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can ...

Dissection of Pelvic Autonomic Ganglia and Associated Nerves in Male and Female Rats

The bilateral major pelvic ganglia (MPG; synonym, pelvic ganglia) are the primary source of postganglionic sympathetic and parasympathetic neurons innervating pelvic organs of rodents; the functionally equivalent structure in humans is the inferior hypogastric plexus. The major pelvic ganglia also provide the route by which lumbar and sacral sensory axons reach the pelvic organs. These complex, mixed ganglia can prove challenging to identify and dissect for further experimental study of normal autonomic mechanisms or to establish preclinical models of disease, injury or visceral pain. Here we describe a protocol to access and visualize these ganglia and their associated nerve tracts. We provide this protocol with schematics for both male and female rats, as the ganglion size and landmarks for identification differ between sexes. The protocol describes removal of the ganglion for in vitro studies, but this method can be integrated into a surgical recovery protocol for experimental interventions (e.g., nerve crush, nerve resection) or for mapping neuronal circuits (e.g., by microinjection of neural tracers). We also demonstrate the primary structures of the ganglion and its associated nerves immediately following dissection and following immunohistochemical staining.

The major pelvic ganglia contain parasympathetic and sympathetic neurons that innervate pelvic organs. Here we describe a dissection method and provide schematics for identification of these ganglia and their associated nerves. These methods can be applied to experimental manipulation of these ganglia in vivo or removal post-mortem for further study.