Name: an optimized procedure for fluorescence-activated cell sorting (facs) isolation of autonomic neural progenitors from visceral organs of fetal mice
Uploaded: 2012-08-17T13:00:00
Description: 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

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?...

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	<li>Gong, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gene+expression+atlas+of+the+central+nervous+system+based+on+bacterial+artificial+chromosomes.">A gene expression atlas of the central nervous system based on bacterial artificial chromosomes.</a> Nature. 425, 917-925 (2003).</li><li>Morrison, S. J., White, P. M., Zock, C., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+identification,+isolation+by+flow+cytometry,+and+in+vivo+self-renewal+of+multipotent+mammalian+neural+crest+stem+cells.">Prospective identification, isolation by flow cytometry, and in vivo self-renewal of multipotent mammalian neural crest stem cells.</a> Cell. 96, 737-749 (1999).</li><li>Kruger, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+stem+cells+persist+in+the+adult+gut+but+undergo+changes+in+self-renewal,+neuronal+subtype+potential,+and+factor+responsiveness.">Neural crest stem cells persist in the adult gut but undergo changes in self-renewal, neuronal subtype potential, and factor responsiveness.</a> Neuron. 35, 657-669 (2002).</li><li>Bixby, S., Kruger, G. M., Mosher, J. T., Joseph, N. M., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-intrinsic+differences+between+stem+cells+from+different+regions+of+the+peripheral+nervous+system+regulate+the+generation+of+neural+diversity.">Cell-intrinsic differences between stem cells from different regions of the peripheral nervous system regulate the generation of neural diversity.</a> Neuron. 35, 643-656 (2002).</li><li>Walters, L. C., Cantrell, V. A., Weller, K. P., Mosher, J. T., Southard-Smith, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+background+impacts+developmental+potential+of+enteric+neural+crest-derived+progenitors+in+the+Sox10Dom+model+of+Hirschsprung+disease.">Genetic background impacts developmental potential of enteric neural crest-derived progenitors in the Sox10Dom model of Hirschsprung disease.</a> Human Molecular Genetics. , (2010).</li><li>Corpening, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+live+imaging+of+enteric+progenitors+based+on+Sox10-Histone2BVenus+transgene+expression.">Isolation and live imaging of enteric progenitors based on Sox10-Histone2BVenus transgene expression.</a> Genesis. 49, 599-618 (2011).</li><li>Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Isolation of fetal rat neural crest stem cells (NCSC) from gut and sciatic nerve. , (2012).</li><li>Skarnes, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conditional+knockout+resource+for+the+genome-wide+study+of+mouse+gene+function.">A conditional knockout resource for the genome-wide study of mouse gene function.</a> Nature. 474, 337-342 (2011).</li><li>Harding, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GUDMAP+database--an+online+resource+for+genitourinary+research.">The GUDMAP database--an online resource for genitourinary research.</a> Development. 138, 2845-2853 (2011).</li><li>Joseph, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+glia+are+multipotent+in+culture+but+primarily+form+glia+in+the+adult+rodent+gut.">Enteric glia are multipotent in culture but primarily form glia in the adult rodent gut.</a> J. Clin. Invest. 121, 3398-3411 (2011).</li><li>Newgreen, D. F., Murphy, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+cell+outgrowth+cultures+and+the+analysis+of+cell+migration.">Neural crest cell outgrowth cultures and the analysis of cell migration.</a> Methods Mol. Biol. 137, 201-211 (2000).</li></ol>

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 ...

Research

JoVE Journal

Neuroscience

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

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

神经细胞集落形成细胞分析检测，以区分善意的神经祖细胞的神经干细胞

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

科研

神经科学

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

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 ...

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

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

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

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

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

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

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

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

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

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 ...