Name: isolation and culture of adult zebrafish brain-derived neurospheres
Uploaded: 2016-02-29T15:00:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres at JoVE.com

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?...

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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。

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			<td>11330-032</td>
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			<td>L-Cysteine hydrochloride monohydrate&#160;</td>
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			<td>N-2 supplement (100x) liquid&#160;</td>
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			<td>15630</td>
			<td>1M</td>
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			<td>D-(+)-Glucose 45%&#160;</td>
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			<td>G8769</td>
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			<td>Life Technologies</td>
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			<td>Sigma</td>
			<td>I5500-50 mg</td>
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			<td>DNAse</td>
			<td>Sigma</td>
			<td>DN25-10mg</td>
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			<td>Papain&#160;</td>
			<td>Worthington Biochemical Corporation</td>
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			<td>PFA&#160;</td>
			<td>TCI</td>
			<td>P0018</td>
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			<td>PBS</td>
			<td>AmericanBio</td>
			<td>AB11072-04000</td>
			<td></td>
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			<td>Tricaine MS-222</td>
			<td>Sigma</td>
			<td>A5040</td>
			<td>stock solution of 4 mg/ml.&#160;</td>
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			<td>Trycold gel&#160;</td>
			<td>Sigma</td>
			<td>TGP8</td>
			<td>gel pack</td>
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			<td>Amaxa Basic Nucleofector Kit</td>
			<td>Lonza</td>
			<td>VPI-1004</td>
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			<td>Trypan blue stain&#160;</td>
			<td>Life Technologies</td>
			<td>15250061</td>
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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 ...

Research

JoVE Journal

Developmental Biology

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

肾再生的成年斑马鱼庆大霉素性损伤

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

科研

发育生物学

Isolation and Characterization of Single Cells from Zebrafish Embryos

分离和斑马鱼胚胎单细胞的表征

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Isolation and Expansion of Adult Canine Hippocampal Neural Precursors

分离和成人犬海马神经前体扩张

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural ...

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制备及文化活动从成年和老年人类骨骼肌成肌前体细胞/成肌细胞主要的

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting ...

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

成年转基因斑马鱼视网膜外植体培养由多光子显微镜活细胞成像

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

小鼠骨髓基质细胞与骨破骨祖的分离、培养及分化

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

人成体和胎心标本中原发性心外膜细胞的分离培养

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

Culture and Transfection of Zebrafish Primary Cells

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 ...

Nanoparticle-mediated siRNA Gene-silencing in Adult Zebrafish Heart

纳米粒子介导的 siRNA 基因沉默在斑马鱼心脏中的表达

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

成人斑马鱼损伤模型研究泼尼松对再生骨组织的影响

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...