The authors thank Guillermina Hill-Teran and Marie-Elise Schwartz for assistance. This work was supported by grants from the National Institutes of Health (5R00HL105791 to S.N.) and from the Alzheimer (NIRP 12-259162). This work was also supported by Institut National de la Sant&#233; et de la Recherche M&#233;dicale (CFC and JLT), Agence Nationale de la Recherche (13-BSV4-0002-01 (JLT), NIH (1R01EB016629-01A1 (JLT), Connecticut Stem Cell Research Fund (13-SCA-Yale-04 (JLT).

野生 CF株斑马鱼根据由耶鲁大学机构动物护理和使用委员会（IACUC协议号2012-11473）批准的方案提出并保持。所有实验应先通过所有相关的政府和机构伦理规范有关动物用于研究目的机构的批准。 1.准备<ol><li>准备10毫升夹层介质中，加入200微升100X青链霉素成9.8毫升DMEM / F12的。 </li><li>准备L-半胱氨酸溶液:10毫升水进行组织培养，添加120毫克的L-半胱氨酸。储存在4℃的L-半胱氨酸溶液长达2周，或在-20℃下长达1年等分试样。 </li><li>准备DNA酶I溶液:1毫升水进行组织培养，加入10毫克的DNase I.保存在4℃的DNA酶I溶液长达2个月。 </li><li>准备好木瓜蛋白酶溶液:准备木瓜蛋白酶SOLUT离子，添加100μl的木瓜蛋白酶（约140单位），将100μlDNA酶I（1％）和200微升L-半胱氨酸（12毫克/毫升）到5毫升的DMEM / F12的。刚准备木瓜蛋白酶溶液每次和在使用前用0.22微米孔径过滤消毒。 </li><li>制备洗涤溶液:以制备100ml的洗涤溶液中，添加650微升葡萄糖45％，500微升的HEPES的1M和5ml FBS的成93.85毫升DPBS 1倍。消毒在使用前用0.22微米孔径的过滤器的洗涤液。储存在4℃下长达2个月的洗涤溶液。 </li><li>制备胰岛素溶液:以制备加入2ml胰岛素溶液，加10的1N NaOH和100mg胰岛素的25微升落入2ml水进行组织培养。 </li><li>制备EGF和FGF的解决方案:在100微克/毫升浓度溶解在DMEM / F12两者有丝分裂原。存储为在-20℃下10微升等分试样。 </li><li>在-20℃下为500微升和1ml等份，respectiv存储器B-27和N-2补充剂:制备B-27和N-2个媒体伊利。 </li><li>制备的Z-分化条件培养基:向制备100毫升的Z-分化条件培养基中，添加40微升的胰岛素（50毫克/毫升），加入500μl的B-27，将1ml N-2，650微升葡萄糖45％和1毫升100×青链霉素成97.81毫升DMEM / F12的。消毒使用前先用0.22微米孔径过滤器Z-分化状态中。储存在4℃的Z分化条件培养基长达1周。 </li><li>准备Z-条件培养基:准备50毫升Z-条件介质，加入EGF的10微升和10微升的FGF到50ml无菌Z-分化条件介质（20毫微克/毫升）。储存在4℃的Z条件培养基长达1周。 </li><li>准备涂布液分化培养:对5毫升外涂料溶液，加100微升细胞外基质溶液加入到4.9毫升DMEM / F12（ 如基质胶）。在冰上4℃解冻的细胞外基质的解决方案。一旦解冻，保持在4℃下üp来2周。 </li></ol> 2.成年斑马鱼脑解剖<ol><li>准备通过填写100毫米×15毫米的培养皿凝胶冰袋床的夹层。然后放置在培​​养皿的盖子，并在-20℃下孵育直至凝胶冻结。在盖处的顶部干净滤纸的平方和包裹都滤纸和陪替氏培养皿用塑料薄膜。 </li><li>清洁和每次使用前消毒用70％乙醇或热的所有显微切割仪器。将所有消毒清扫工具解剖显微镜附近，安乐死前右方，放置解剖床上光纤照明显微镜下。 </li><li>收集2的全脑神经球准备成年斑马鱼;和3至4斑马鱼产生从解剖脑区的神经球。 </li><li>使用安乐死的机构动物护理和使用委员会批准的方案成年斑马鱼（8-12个月）。接下来，浸泡的鱼7为5-10秒5％的乙醇和快速地放置在解剖床随后断头在使用手术刀鳃的水平。 <ol><li>安乐死的动物，施用过量三卡因甲磺酸（300毫克/升），直到动物的心脏搏动逐渐减慢和循环停止，然后浸入冰水。 </li></ol></li><li>转动头部背面向下，并用剪刀从做切口侧到嘴的纵向切割。使用镊子暴露颅底并删除所有邻近组织。从朝向顶盖脊髓的开头削减头骨的侧壁。 </li><li>使用剪刀，切割并除去视神经，然后在顶盖的水平除去头骨的最外侧的部分的两侧上。转动头部腹面朝上。使用镊子，剥离头骨的最前端部的其余部分。 </li><li>与颅骨剩余部分转移大脑一起到一个新菜的WiTH的夹层介质（1.1）。使用微刀塑料处理干净的夹层介质脑组织，保持所有大脑结构完好。 </li><li>从这一点来说，使用全斑马鱼脑继续的协议。 <ol><li>或者适应此协议特定脑区产生从整个斑马鱼大脑或从一个新鲜的手术刀解剖端脑，顶盖/间脑小脑或神经球。使用神经特异性荧光斑马鱼神经转基因系解剖根据感兴趣的大脑区域图3和附图11。 </li></ol></li></ol>成人脑3.单细胞分离<ol><li>在生物安全柜进行所有的后续工作。转移的脑组织成含有800微升夹层介质的1.5ml试管（在步骤1.1中制备）。通过移液除去夹层介质，而不触及脑组织。 </li><li>加入500微升的木瓜蛋白酶溶液（步骤1.4）和消化在37℃下脑组织10分钟。不要孵育时间超过15分钟，因为它可以降低细胞活力。 </li><li>温育后，用1000微升吸管尖切从底部〜0.25英寸转移用500μl的木瓜蛋白酶溶液的脑组织入15ml锥形管中。通过用相同的尖端缓慢地上下吹打10倍轻轻解离脑组织。不吸管上下15倍以上，并在此步骤中不产生气泡，因为它可能改变细胞生存力。 </li><li>在37℃下再孵育脑组织10分钟，随后吹吸，并使用未切割1000微升定期尖端向下10-13倍。不吸管上下15倍以上，以避免细胞死亡。 </li><li>通过加入2ml洗涤液（步骤1.5），并离心细胞悬浮液以800×g离心，在室温（RT）下5分钟，停止酶消化。小心倒出标upernatant并通过与1000微升定期尖大力用手指，然后轻敲管通过上下吹打悬浮在剩余溶液中的沉淀。接着，加入2毫升的洗涤溶液。 </li><li>在这个阶段，检查已经获得单细胞悬浮液，在显微镜下。在800 xg离心再次离心细胞悬浮液在室温下5分钟。去除上清，重新悬浮，用1毫升新鲜Z-条件培养基（步骤1.10）的颗粒。 </li><li>染色细胞通过使用台盼蓝12和使用血球排除蓝死细胞计数的活细胞。制备与细胞悬浮液的200微升和300微升新鲜的Z-条件培养基在每个孔24孔板中。种子细胞以〜500个细胞/微升的密度。保持在培养箱中培养细胞在30℃下在5％CO 2中 ，由于斑马鱼脑源性神经球不在37℃下生长良好。 </li></ol> 4.原代神经球&lt;/ P&gt; <ol><li>后第1天的体外 （DIV1），观察从显微镜下的成人全脑得到的单细胞悬浮液，并注意碎片是否已经在井的中心积累。仔细吹打约100微升介质的碎屑（少如果可能的话）。然后加入100微升新鲜Z-条件培养基。单细胞悬浮液应在此时（ 图2A DIV1）被观察到。 </li><li>经过体外培养 2天（DIV2），扩大培养的细胞。使用带尖砍1000微升吸管，收集并从每孔转移250微升细胞悬液到一个新的好。加入250微升新鲜Z-条件培养基至所有孔。扩大培养细胞之前，在整个井轻轻地均质化悬浮液通过上下抽吸。 </li><li>重复步骤体外以下4天（DIV4）4.1和4.2。在神经球的大小逐步增加应该是六sible在中心和阱的边缘在DIV3和DIV4（ 图2B和C）。 注:主神经球可通行或分化评估的多能性进行处理。可替代地，他们可以为核转染或脂转染11被加工在分化过程来表征特定基因的作用。 </li></ol> 5.传代初级神经球<ol><li>从每个孔中取出250μl的组织培养和机械解离DIV4神经球以1毫升吸管。不要吸管上下过快空气形成气泡可以增加细胞死亡。 </li><li>计数与血球细胞和250μl的主培养物上清液的分发800细胞/微升到24孔板的每个孔中。 </li><li>加入250微升的Z-条件培养基的各孔中。 </li><li>经过体外培养 2天（DIV2），扩大培养的细胞在步骤4.2报告ð4.3。 </li></ol> 6.主要分化的神经球<ol><li>通过浸没消毒盖玻片在70％乙醇10分钟，然后干燥盖玻片在RT将它们放置在一个12孔板的每个孔中。 </li><li>添加300μl的在玻璃盖的中心外涂布液（步骤1.11）以这样的方式，它可以广泛地扩展，并覆盖整个玻璃盖。然后，孵育在37℃1小时（使用加湿细胞培养孵育器）。 </li><li>除去温育后的外涂层溶液，并在37℃下干燥该玻璃盖2小时。 </li><li>从每个孔中取出250微升的组织文化。机械分离每孔吹打上下所有DIV4主要神经球用1毫升吸管（与更广泛的高端尖）。 </li><li>种子在4×10 3个细胞/ ml在预先制备的外涂覆基质盖玻片的细胞密度（步骤6.1-6解离神经球细胞悬浮液。3）和在5％CO 2保持至少30分钟，在30℃下，直到附着到衬底。然后，除去培养基，并在5％CO 2在30℃下培养细胞24小时前迅速加入1ml预热新鲜的Z-分化条 ​​件培养基（步骤1.9）的。 </li><li>每隔两天，在细胞分化的时间更换一半的Z-分化的条件培养基。 </li></ol> 7.初级神经球的基因操作<ol><li>通过在4℃下在80×g下离心8分钟收集DiV3-4初级神经球（步骤4.3）。准备用于商业oligonucleoatides的脂质体转染如previouly描述11或DNA质粒载体的细胞核-转染。 </li><li>在4×10 3个细胞/微升的在100μl的反应混合物（82微升nucleofactor溶液和18微升补充的）的细胞密度重悬神经球丸。 </li><li>混合神经球suspensi与5微升（1微克/微升质粒股票）所选择的DNA质粒载体。神经球/ DNA混合物转移到一个nucleofector反应杯为nucleotransfection和根据由nucleofector试剂盒中提供的制造商的说明选择nucleofector程序。 </li><li>核转染后，立即加入500ml的预热的Z-条件培养基（1.10）的体积至细胞和板转染的神经球为研究其细胞更新和/或分化特性，如上所述。 </li></ol>

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The authors declare no competing financial interests.

该协议的主要目的是分离和培养成年斑马鱼脑源性神经球用于研究神经干/祖细胞的细胞和分子的特性。在这里，我们报告如何选择多能神经细胞并产生三个神经细胞类型， 即 ，星形胶质细胞，神经元和少突胶质细胞，从成年斑马鱼大脑。因为可重现神经球形成测定法尚未在斑马鱼建立迄今为止协议是非常显著。 在协议中的几个关键步骤，需要得到尊重，并在故障排?...

哺乳动物神经干细胞（NSCs）都被他们在自由浮动的文化成长为划分称为神经球1细胞群的能力，其特征体外 。在表皮生长因子（EGF）和成纤维细胞生长因子（FGF）的存在下，神经干细胞分化或对称，产生自我更新的神经干细胞，或不对称，以产生两个不同的子细胞， 即 ，微分祖细胞和新颖NSC。因此，神经干文化是神经干/祖细胞多分化的神经细胞2-4的混合物的神经干细胞可以，但是，从其他神经干细胞类型由两个特定的属性区别:他们自由显示屏长期自我更新浮培养物，它们可以分化成以下的生长因子撤出和粘附到细胞外基质底物的所有神经细胞谱系（ 即 ，神经元，星形胶质细胞和少突胶质）。在mammALS的神经球培养系统是用来证明神经干细胞在成人大脑中存在的第一个体外系统和保持来分析增殖，自我更新能力和神经干细胞和祖细胞的多能的最常用的工具。因此，即使球形成测定法从一些缺点和局限性4苦，该培养系统，是从它的体内利基4中取出时，并一直工具在确定关键调节评估细胞的表现为干细胞的潜在有价值NSC的自我更新和细胞命运决定5-7。 相较于哺乳动物谁限制了成年神经，斑马鱼组成产生沿着整个生命全脑轴新的神经细胞。斑马鱼的成人脑显示多个神经源性龛窝藏神经干/祖细胞使斑马鱼强大的模式生物的的理解了他干在脑细胞活动，以及中枢神经系统的再生所需要的分子的程序。在过去的17年里，几个研究小组开发的方法进行分离和培养斑马鱼的神经细胞8,9。这些研究的目的是生产胚胎神经元和在体外神经胶质细胞，而不是在保持NSCs的并调查它们的属性。虽然神经球已在成年Apteronotus leptorhynchus（布朗鬼西刀鱼）10产生的，在斑马鱼神经球形成测定法仍有待建立。 这里，我们描述一个神经球形成测定法来证明斑马鱼神经11中的miR-107的作用。该协议使得:1）无论是从斑马鱼全脑或从几个解剖大脑区域，如端脑，所述顶盖和小脑成人神经干/祖细胞的收集; 2）FL的代浮动和成人神经干/祖细胞的自我更新的神经球; 3）向下和上调编码基因或小的非编码RNA 11在神经球，的表达为了研究它们在神经干/祖细胞的增殖和分化的作用。

<table><tbody><tr><td>DPBS 1x</td><td>Life Technologies</td><td>14190-144</td><td></td></tr><tr><td>DMEM/F12 1x</td><td>Life Technologies</td><td>11330-032</td><td></td></tr><tr><td>L-Cysteine hydrochloride monohydrate&amp;nbsp;</td><td>Sigma</td><td>C6852-25g</td><td></td></tr><tr><td>B-27</td><td>Life Technologies</td><td>17504-044</td><td></td></tr><tr><td>N-2</td><td>Life Technologies</td><td>17502-048</td><td>N-2 supplement (100x) liquid&amp;nbsp;</td></tr><tr><td>HEPES&amp;nbsp;</td><td>Life Technologies</td><td>15630</td><td>1 M</td></tr><tr><td>D-(+)-Glucose 45%&amp;nbsp;</td><td>Sigma</td><td>G8769</td><td></td></tr><tr><td>Penicillin-streptomycin&amp;nbsp;</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>Life Technologies</td><td>16000044</td><td></td></tr><tr><td>Human FGF-basic&amp;nbsp;</td><td>Peprotech</td><td>&amp;nbsp;100-18B</td><td></td></tr><tr><td>Human EGF&amp;nbsp;</td><td>Peprotech</td><td>AF-100-15</td><td></td></tr><tr><td>Insulin&amp;nbsp;</td><td>Sigma</td><td>I5500-50 mg</td><td></td></tr><tr><td>DNAse</td><td>Sigma</td><td>DN25-10mg</td><td></td></tr><tr><td>Papain&amp;nbsp;</td><td>Worthington Biochemical Corporation</td><td>LS003126</td><td></td></tr><tr><td>Matrigel&amp;nbsp;</td><td>Becton Dickinson</td><td>356234</td><td></td></tr><tr><td>PFA&amp;nbsp;</td><td>TCI</td><td>P0018</td><td></td></tr><tr><td>PBS</td><td>AmericanBio</td><td>AB11072-04000</td><td></td></tr><tr><td>Tricaine MS-222</td><td>Sigma</td><td>A5040</td><td>stock solution of 4 mg/ml.&amp;nbsp;</td></tr><tr><td>Trycold gel&amp;nbsp;</td><td>Sigma</td><td>TGP8</td><td>gel pack</td></tr><tr><td>Amaxa Basic Nucleofector Kit</td><td>Lonza</td><td>VPI-1004</td><td></td></tr><tr><td>Trypan blue stain&amp;nbsp;</td><td>Life Technologies</td><td>15250061</td><td></td></tr></tbody></table>

isolation and culture of adult zebrafish brain-derived neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.

在成年斑马鱼神经球文化的总体方案在这里，我们描述了从成年斑马鱼脑进行神经球形成测定法的协议的所有步骤。首先，成人神经干/祖细胞已经或者从斑马鱼全脑或从几个解剖大脑区域，如端脑，所述顶盖和小脑（ 图1A-C）的收集。成人神经干/祖细胞的单细胞悬浮液已然后被用来产生浮动和自我更新的神经球（...

Watch this Scientific Journal Video about Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres at JoVE.com

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

在这里，我们提供一个可重复的方法，使用从整个脑或从任一端脑，顶盖或成年斑马鱼脑的小脑区域衍生的神经球测定以检查成人神经发生。此外，我们描述来操纵在斑马鱼神经球的基因表达的步骤。

分离培养成年斑马鱼脑源性神经球

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration ...

isolation-and-culture-of-adult-zebrafish-brain-derived-neurospheres

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

12.9K 次观看.  Yale University. 在这里，我们提供一个可重复的方法，使用从整个脑或从任一端脑，顶盖或成年斑马鱼脑的小脑区域衍生的神经球测定以检查成人神经发生。此外，我们描述来操纵在斑马鱼神经球的基因表达的步骤。 

视频: Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

成年鼠神经干细胞的分离和扩大使用神经干含量

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

科研

神经科学

Culture and Transfection of Zebrafish Primary Cells

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in an intact and developing vertebrate. However, the detailed imaging of the morphologies of distinct cell types and subcellular structures is limited in whole mounts. Therefore, we established an efficient and easy-to-use protocol to culture live primary cells from zebrafish embryos and adult tissue.
In brief, 2 dpf zebrafish embryos are dechorionated, deyolked, sterilized, and dissociated to single cells with collagenase. After a filtration step, primary cells are plated onto glass bottom dishes and cultivated for several days. Fresh cultures, as much as long term differenciated ones, can be used for high resolution confocal imaging studies. The culture contains different cell types, with striated myocytes and neurons being prominent on poly-L-lysine coating. To specifically label subcellular structures by fluorescent marker proteins, we also established an electroporation protocol which allows the transfection of plasmid DNA into different cell types, including neurons. Thus, in the presence of operator defined stimuli, complex cell behavior, and intracellular dynamics of primary zebrafish cells can be assessed with high spatial and temporal resolution. In addition, by using adult zebrafish brain, we demonstrate that the described dissociation technique, as well as the basic culturing conditions, also work for adult zebrafish tissue.

We present an efficient and easy-to-use protocol for preparing primary cell cultures of zebrafish embryos for transfection and live cell imaging as well as a protocol to prepare primary cells from adult zebrafish brain.

斑马鱼原代细胞的培养与转染

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in ...

发育生物学

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 ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

分离和成人神经干细胞的培养从鼠标Subcallosal区

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

斑马鱼脑幼虫神经元、巨噬细胞和小胶质的分离和 RNA 提取

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

Culture of Neurospheres Derived from the Neurogenic Niches in Adult Prairie Voles

Neurospheres are primary cell aggregates that comprise neural stem cells and progenitor cells. These 3D structures are an excellent tool to determine the differentiation and proliferation potential of neural stem cells, as well as to generate cell lines than can be assayed over time. Also, neurospheres can create a niche (in vitro) that allows the modeling of the dynamic changing environment, such as varying growth factors, hormones, neurotransmitters, among others. Microtus ochrogaster (prairie vole) is a unique model for understanding the neurobiological basis of socio-sexual behaviors and social cognition. However, the cellular mechanisms involved in these behaviors are not well known. The protocol aims to obtain neural progenitor cells from the neurogenic niches of the adult prairie vole, which are cultured under non-adherent conditions, to generate neurospheres. The size and number of neurospheres depend on the region (subventricular zone or dentate gyrus) and sex of the prairie vole. This method is a remarkable tool to study sex-dependent differences in neurogenic niches in vitro and the neuroplasticity changes associated with social behaviors such as pair bonding and biparental care. Also, cognitive conditions that entail deficits in social interactions (autism spectrum disorders and schizophrenia) could be examined.

We established the conditions to culture neural progenitor cells from the subventricular zone and dentate gyrus of the adult brain of prairie voles, as a complementary in vitro study, to analyze the sex-dependent differences between neurogenic niches that could be part of functional plastic changes associated with social behaviors.

从成人草原卷的神经性尼奇衍生的神经球培养

Neurospheres are primary cell aggregates that comprise neural stem cells and progenitor cells. These 3D structures are an excellent tool to determine the ...

<meta charset="utf8"></head><body>对从小鼠脑室下区分离的 NSC 进行高效成核

Isolation, Expansion, and Nucleofection of Neural Stem Cells from Adult Murine Subventricular Zone

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) as mitogens, producing clonal aggregates known as neurospheres. This in vitro system is a valuable tool for studying NSC potential. Transfection of siRNAs or genes carried in plasmids can be used to induce perturbations to gene expression and study NSC biology. However, the exogenous nucleic acid delivery to NSC cultures is challenging due to the low efficiency of central nervous system (CNS) cells transfection. Here, we present an improved nucleofection system that achieves high efficiency of gene delivery in expanded NSCs from adult murine SVZ. We demonstrate that this relatively simple method enhances gene perturbation in adult NSCs, surpassing traditional transfection protocols with survival rates exceeding 80%. Moreover, this method can also be applied in primary isolated NSCs, providing a crucial advancement in gene function studies through gene expression manipulation via knockdown or overexpression in neurosphere cultures.

<meta charset="utf8"></head><body>作者聚焦：改进的核转染技术，在小鼠脑室下区衍生的神经干细胞培养物中实现高效基因递送

Here, we describe a nucleofection system designed to enhance gene delivery efficiency in expanded neural stem cells (NSCs) isolated from the adult murine subventricular zone. The findings demonstrate that this method significantly improves gene perturbation in NSCs, surpassing the effectiveness of traditional transfection protocols and enhancing cell survival rate.

来自成体小鼠脑室下区的神经干细胞的分离、扩增和成核转染

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented ...

Generation and Differentiation of Primary Neurospheres from Zebrafish Neural Stem Cells

Source: Lopez-Ramirez, M. A. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/53617">Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres.</a>&#160;J. Vis. Exp. (2016)
This video demonstrates the generation and differentiation of primary neurospheres. Neural stem cells from zebrafish, in the presence of growth factors, proliferate into free-floating neurospheres with self-renewal capacity, which later differentiate into glial cells and neurons.

斑马鱼神经干细胞原代神经球的产生和分化

1. Preparations

	Prepare 10 ml of dissection medium, and add 200 &#181;l of 100x penicillin-streptomycin into 9.8 ml of Dulbecco&#39;s Modified Eagle ...

分离培养成年斑马鱼脑源性神经球

摘要

探索更多视频

此视频中的章节

成年鼠神经干细胞的分离和扩大使用神经干含量

斑马鱼原代细胞的培养与转染

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

分离和成人神经干细胞的培养从鼠标Subcallosal区

斑马鱼脑幼虫神经元、巨噬细胞和小胶质的分离和 RNA 提取

从成人草原卷的神经性尼奇衍生的神经球培养

来自成体小鼠脑室下区的神经干细胞的分离、扩增和成核转染

来自成体小鼠脑室下区的神经干细胞的分离、扩增和成核转染

来自成体小鼠脑室下区的神经干细胞的分离、扩增和成核转染

斑马鱼神经干细胞原代神经球的产生和分化