Name: isolation and purification of drosophila peripheral neurons by magnetic bead sorting
Uploaded: 2009-12-01T14:59:00
Description: Watch this Scientific Journal Video about Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting at JoVE.com

我们感谢博士。裕农一月和韦斯Grueber提供在这项研究中所使用的飞股票。作者承认托马斯F和支持这项研究的凯特米勒Jeffress纪念信托（DNC）和乔治梅森大学教务办公室（EPRI）。

果蝇外周神经元的磁性珠排序总评（共完成“议定书”的时间：2.5-3小时） 在任何时候都保持一个干净，核糖核酸酶自由的环境标准的实验室程序必须遵守，以防止RNA降解。 当解剖果蝇幼虫角质层，并放置在细胞内分解缓冲区，外周神经元是脱离角质层的细胞之一。我们必须利用这个属性，并设计了这个协议，以去除大部分从非特异性的细...

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	<li>Corty, M. M., Matthews, B. J., Grueber, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecules+and+mechanisms+of+dendrite+development+in+Drosophila.">Molecules and mechanisms of dendrite development in Drosophila.</a> Development. 136, 1049-1061 (2009).</li><li>Parrish, J. Z., Emoto, K., Kim, ., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+that+regulate+establishment,+maintenance,+and+remodeling+of+dendritic+fields.">Mechanisms that regulate establishment, maintenance, and remodeling of dendritic fields.</a> Ann. Rev. Neurosci. 30, 399-423 (2007).</li><li>Lee, T., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaic+analysis+with+a+repressible+cell+marker+for+studies+of+gene+function+in+neuronal+morphogenesis.">Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis.</a> Neuron. 22, 451-461 (1999).</li><li>Grueber, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projections+of+Drosophila+multidendritic+neurons+in+the+central+nervous+system:+links+with+peripheral+dendrite+morphology.">Projections of Drosophila multidendritic neurons in the central nervous system: links with peripheral dendrite morphology.</a> Development. 134, 55-64 (2007).</li><li>Song, W., Onishi, M., Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+multidendritic+sensory+neurons+are+necessary+for+rhythmic+locomotion+behavior+in+Drosophila+larvae.">Peripheral multidendritic sensory neurons are necessary for rhythmic locomotion behavior in Drosophila larvae.</a> Proc Natl Acad Sci USA. 104, 5199-5204 (2007).</li><li>Livak, K. J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2(-Delta+Delta+C(T))+methods.">Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) methods.</a> Methods. 25, 402-408 (2001).</li></ol>

这里介绍的协议优化，坚持紧紧地使用磁珠细胞分选策略果蝇三龄幼虫角质层内表面的外周神经元的分离和纯化。虽然我们已经使用了此协议专门隔离果蝇DA能神经元，此协议的应用到其他类型的细胞，角质层中的幼虫或蛹的发展阶段，坚持以隔离（如上皮细胞，肌肉，其他的外周神经元）可适应由不同的几个参数，并使用不同的GAL4，记者UAS - mCD8 - GFP转基因标签的细胞类型或类型的利益。...

设备<ul><li> （10）钕块磁铁（K＆J磁公司，卡特彼勒。B444B），或者DynaMag™-2（Invitrogen公司，卡特彼勒。＃123 - 21D），也可使用。 </li><li> Kontes玻璃组织研磨机，2毫升的工作能力，与大间隙杵（货号＃885300-0002） </li><li>细胞过滤器，如磁珠预分离过滤器（美天旎生物技术公司，卡特彼勒＃130-041-407） </li><li> 35毫米培养皿菜涂有Sylgard（道康宁公司，卡特彼勒＃3097358-1004） </li><li>解剖工具：两对杜蒙号5钳（精细的科学工具，猫＃11251-20。） </li><li> Vannas春天微解剖剪刀（精细的科学工具，猫＃15000-08） </li><li>配有一次性尖头滴管（P - 1000 P - 100 P - 10） </li><li>标准血球计数细胞和估计纯度</li><li>与标准直径的火抛光提示巴氏吸管〜50％的标准直径，缩小和收窄至〜25％的标准直径为trituration </li><li>台面微型离心机（1-16,000（X）G） </li><li>涡旋</li><li>荧光立体显微镜（徕卡MZ16FA在本议定书） </li></ul>

isolation and purification of drosophila peripheral neurons by magnetic bead sorting

“<em&gt;果蝇</em&gt;周围神经系统（PNS）是一个强大的调查功能和分子水平的神经元发育和树突形态发生的复杂过程模型。为了帮助这些分析，我们已经开发出一种称为树突分枝（DA）神经元，已被广泛用于研究树突形态发生的PNS的神经元的一个子类的隔离策略<sup&gt; 1,2</sup&gt;。这些神经元是非常困难的隔离作为一个纯粹的人口，部分原因是其极低的发生，他们难以达到低于强硬的几丁质的幼虫角质层位置。我们新开发的方法克服了这些挑战，是一个快速和特定的细胞抗体包被的磁珠富集。磁珠分选的研究，我们已经使用了年龄相仿的三龄幼虫，表达了小鼠CD8标记的GFP融合蛋白（<em&gt;无人机mCD8 - GFP</em&gt;）<sup&gt; 3</sup&gt;控制下的IV级树突分枝（DA）神经元特异性<em&gt;扒手（PPK）- GAL4</em&gt;驱动程序<sup&gt; 4</sup&gt;或控制的泛- DA神经元特异性GAL4<sup&gt; 21-7</sup&gt;驱动程序<sup&gt; 5</sup&gt;。虽然此协议已被隔离PNS的不同的几个参数，连接到幼虫角质层的内壁细胞优化，可以使用相同的协议，以分离出许多不同的细胞类型，幼虫或蛹角质层发展（如上皮细胞，肌肉，oenocytes等），或从GAL4特定的驱动程序的表达模式后的幼虫机关的其他类型的细胞。用这种方法分离出的RNA质量高，可以很容易地用于下游基因组分析，如微阵列基因表达分析研究。这种方法提供了一个强大的新工具来进行孤立的研究<em&gt;果蝇</em&gt;树突分枝（DA）神经元底层树突形态发生的分子机制，从而提供了新的见解。

Watch this Scientific Journal Video about Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting at JoVE.com

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

在这个视频文章中，我们目前的分离纯化的方法<em&gt;果蝇</em&gt;使用快速磁珠辅助细胞排序策略外周神经元。从分离的细胞获得的RNA可以很容易地用于下游应用，包括微阵列分析。

分离纯化果蝇</em磁性微球的排序>外周神经元

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Research

JoVE Journal

Biology

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

科研

生物学

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

由不稳定的多蛋白质复合物的分离在体内控制细胞的交联和免疫磁性亲和层析

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Isolation of Drosophila melanogaster Testes

Isolation of Drosophila melanogaster Testes

分离果蝇睾丸

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Isolation and Purification of Kinesin from Drosophila Embryos

Isolation and Purification of Kinesin from Drosophila Embryos

kinesin的分离和纯化果蝇胚胎

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Chromatin Isolation by RNA Purification (ChIRP)

Chromatin Isolation by RNA Purification ChIRP

RNA纯化的染色质隔离（CHIRP）

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

新鲜组织分离正常和癌症相关的成纤维细胞的荧光激活细胞分选（FACS）

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

Purification of Transcripts and Metabolites from Drosophila Heads

Purification of Transcripts and Metabolites from Drosophila Heads

成绩单和代谢产物的分离纯化<em&gt;果蝇</em&gt;头

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

细胞器运输的培养<em&gt;果蝇</em&gt;细胞：S2细胞系和原代神经元。

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

鉴定蛋白质 - 蛋白质相互作用的<em&gt;果蝇</em&gt;成人头由串联亲和纯化（TAP）

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

分离纯化果蝇外周神经元