Name: primary microglia isolation from mixed glial cell cultures of neonatal rat brain tissue
Uploaded: 2012-08-15T12:00:00
Description: Watch this Scientific Journal Video about Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue at JoVE.com

在统一服务大学的校内计划资助。

1。新生大鼠脑组织解剖<ol><li>寒意莱博维茨的L-15条件培养基（莱博维茨的L-15 + 0.1％BSA + 1％笔/ STREP）至4°C。温暖的培养基（DMEM培养基+ 10％胎牛血清+ 1％青霉素/链霉素）至37°C。准备4-5毫升条件的L-15冰媒体4 60x15毫米培养皿。 100％的酒精消毒所有的手术工具。 </li><li>用70％乙醇冲洗P2新生儿说唱幼仔。 （P1〜P5之间的新生大鼠岁的幼崽，可用于在该协议）。 </li><li>迅速斩首老鼠幼犬用无菌锋利?...

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+of+highly+enriched+and+quiescent+microglia+from+adult+rat+hippocampus:+immunophenotypic+and+functional+characteristics.">Rapid isolation of highly enriched and quiescent microglia from adult rat hippocampus: immunophenotypic and functional characteristics.</a> J. Neurosci. Methods. 151, 121-130 (2006).</li><li>Byrnes, K. R., Stoica, B., Loane, D. J., Riccio, A., Davis, M. I., Faden, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabotropic+glutamate+receptor+5+activation+inhibits+microglial+associated+inflammation+and+neurotoxicity.">Metabotropic glutamate receptor 5 activation inhibits microglial associated inflammation and neurotoxicity.</a> Glia. 57, 550-560 (2009).</li><li>Loane, D. J., Stoica, B. 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虽然此协议经常用来生产纯净和健康的小胶质细胞的研究实验，在程序的关键部件的技术方面的慎重考虑，将尽量减少在孤立的小胶质细胞的变化。首先，是要尽量减少缺氧和缺血性脑损伤的组织，在新生幼鼠脑组织工作，及时清扫。然而，同样重要的是从大脑中彻底清除脑膜覆盖在解剖，因为在场的脑膜将有助于显着的成纤维细胞污染，由于这些培养条件下的快速增殖率。 <p class="jove_content"...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> 60毫米×15毫米培养皿</td><td>费希尔品牌</td><td> 0875713A </td><td></td></tr><tr><td>夏普解剖剪刀</td><td>罚款科学工具</td><td> 14094-11 </td><td></td></tr><tr><td>杜蒙＃7B钳标准提示，弯曲，11厘米</td><td>罚款科学工具</td><td> 11270-20 </td><td></td></tr><tr><td>杜蒙＃5镊子标准的提示，直，11厘米</td><td>罚款科学工具</td><td> 11251-10 </td><td></td></tr><tr><td> 50毫升锥形离心管</td><td>厂商VWR </td><td> 89039-656 </td><td></td></tr><tr><td> 5毫升血清移液器</td><td>格雷尼尔生物一</td><td> 606180 </td><td></td></tr><tr><td> 10毫升血清移液器</td><td>格雷尼尔生物一</td><td> 607180 </td><td></td></tr><tr><td> 100微米无菌尼龙细胞过滤器</td><td>鹘</td><td> 35-2360 </td><td></td></tr><tr><td> 75厘米2组织培养瓶</td><td>康宁</td><td> 430641 </td><td></td></tr><tr><td> Dulbecco的最低基本培养基（DMEM培养基） </td><td> Gibco公司（Invitrogen公司） </td><td> 31053-028 </td><td></td></tr><tr><td>莱博维茨的L-15培养基</td><td> Gibco公司（Invitrogen公司） </td><td> 11415064 </td><td></td></tr><tr><td>胎牛血清</td><td> Gibco公司（Invitrogen公司） </td><td> 16000-036 </td><td></td></tr><tr><td>胎儿马血清</td><td>费舍尔</td><td> SH3007402 </td><td></td></tr><tr><td>青霉素链霉素</td><td> Gibco公司（Invitrogen公司） </td><td> 15140163 </td><td></td></tr><tr><td> 100％的乙醇</td><td>华纳格雷厄姆公司</td><td> 64-17-5 </td><td></td></tr><tr><td>磷酸盐缓冲盐溶液，10X，pH值7.4 </td><td>质量生物学，INC。 </td><td> 119-069-131 </td><td> 1X在无菌蒸馏水</td></tr><tr><td>生化危机袋</td><td>厂商VWR </td><td> 14220-028 </td><td></td></tr><tr><td>血球</td><td>豪塞尔科学</td><td> 1492 </td><td></td></tr><tr><td> 6井细胞与cellBIND表面培养皿</td><td>康宁</td><td> 3335 </td><td></td></tr></tbody></table>

primary microglia isolation from mixed glial cell cultures of neonatal rat brain tissue

小胶质细胞在哺乳动物的脑细胞的总人口约12％的帐户。虽然神经元和神经胶质细胞被认为是神经系统的主要类型的细胞，小胶质细胞发挥 ​​显著作用，在正常脑组织的生理组织碎片和病原体监测和维持动态平衡通过吞噬活性1,2实质。小胶质细胞被激活过程中的损伤和疾病的条件，包括神经退行性疾病，脑外伤，中枢神经系统感染3。这些活化条件下，增加小胶质细胞的吞噬活性，经过morpohological和增殖的变化，积极分泌活性氧和氮物种，促炎性趋化因子和细胞因子，往往激活旁分泌或自分泌环4-6。由于这些小胶质细胞反应在神经系统疾病的发病机制的研究主要集中在microglia保证。 由于大脑细胞的异质性，这是技术上的困难，具有纯度高，在体内实验中获得足够的小胶质细胞的样品材料。小胶质细胞的神经保护和神经功能的研究现状，需要常规的技术方法始终产生足够的产量为研究的纯洁和健康的小胶质细胞。我们提出，在文字和视频，从混合胶质文化的隔离纯初级小胶质细胞的各种下游应用协议。简单地说，这种技术采用分离新生幼鼠脑组织生产混合胶质细胞培养。混合胶质细胞培养达到汇合后，小胶质细胞主要是机械分离的文化由摇晃的时间很短。小胶质细胞，然后镀上高纯度的实验研究。 这种方法的原理和协议在文献7,8所述。此外，隔离初级小胶质细胞的替代方法有很好的描述9-12。匀浆脑组织可通过密度梯度离心分离，产生初级小胶质细胞12。然而，离心是适中的长度（45分钟），并可能会导致细胞损伤和激活，以及，导致富含胶质细胞和其他细胞群。另一个协议已动用隔离初级小胶质细胞的各种生物通过长时间的晃动，而在文化9-11（16小时）。摇动后，媒体上清离心分离小胶质细胞。这不再两步分离方法也可能干扰小胶质细胞的功能和激活。我们主要是利用以下的小胶质细胞的分离协议在我们的实验室有许多原因：（1）初级小胶质细胞在体内的生物学模拟更忠实，比永生的灭鼠小胶质细胞株（2）额定机械破坏，最大限度地减少潜在的细胞功能障碍或激活，（3）没有足够的产量可以通过混合胶质细胞培养获得。 重要的是要注意该协议使用新生幼鼠脑组织小胶质细胞分离和使用旧的大鼠，分离小胶质细胞可以显着影响产量，激活状态，孤立的小胶质细胞的功能特性。有证据表明，老龄化与小胶质细胞功能障碍，增加神经发炎和神经退行性病变的联系，所以以前的研究都采用体外成人小胶质细胞小胶质细胞在神经退行性疾病中的作用更好地了解地方老龄化是一个重要参数。但是，小胶质细胞体外不能被保存在长时间的文化。因此，虽然该协议扩展了初级小胶质细胞中的文化生活，也应注意到，小胶质细胞的行为ðifferently从成人的胶质细胞和体外研究时，应慎重考虑翻译体内设置。

Watch this Scientific Journal Video about Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue at JoVE.com

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

隔离初级小胶质细胞的大脑细胞的异质性是必不可少的，以调查他们在生理和病理条件下的作用。本协议描述了机械隔离和混合细胞培养技术，提供高产量和高纯度的，可行的初级小胶质细胞<em&gt;在体外</em&gt;学习和下游应用。

初级小胶质细胞的分离，从新生大鼠脑组织胶质细胞混合文化

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

Research

JoVE Journal

Immunology and Infection

Isolation of Brain-infiltrating Leukocytes

脑浸润白细胞的分离

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

科研

免疫与感染

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

一种简单而有效的隔离混合原代培养成人肝细胞巨噬细胞的方法

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

从小学的人角质形成细胞器官筏文化的代

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

培养小胶质细胞的新生和成年的中枢神经系统

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

从人类胸腺髓样树突状细胞和上皮细胞的分离

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

皮层小胶质细胞的分离与保存免疫表型和功能从新生儿鼠

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Neuropathogenic的非侵入型<em&gt;大肠杆菌</em&gt;感染乳鼠

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

隔离和小鼠骨髓的静脉注射单核细胞衍生

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

从大鼠睾丸支持细胞的分离和管周细胞

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...