Name: isolation of distinct cell populations from the developing cerebellum by microdissection
Uploaded: 2014-09-21T15:00:00
Description: Watch this Scientific Journal Video about Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection at JoVE.com

作者要感谢詹姆斯Oesterling流式细胞仪的帮助。这项研究是由美国国家癌症研究所（R01-CA178380，ZY）和美国国家研究院​​博士后培训补助（5T32CA009035-37，LWY）的资金支持。

已按照批准的Fox Chase癌症中心的动物护理和使用委员会的程序在执行该协议利用动物。 1，准备工具，解决方案和盖玻片<ol><li>清洁两套＃5细镊子，一组7号细弯钳，一个大的手术剪刀，1 microdissecting剪一小铲和一个带孔的勺子。将仪器的自密封消毒袋和高压锅。请眼袋仪器，直到准备使用。 </li><li>制备3％低熔点琼脂糖溶液。 <ol><li>添加1.5克琼脂糖至50毫升无菌贝科的磷酸盐...

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此处所描述的显微切割的方法是能够分离的活细胞中的分子和功能分析使用的纯群体的第一个过程。这表现在Li 等人9， 通过该方法得到的NEP可以培养并掺入了各种实验，并由于其高纯度的，也可用于微阵列分析。 花时间去精通这种显微切割技术，这是非常重要的。的NEP位于EGL的深区域中的非常接近的分子层，其中巢蛋白表达伯格曼胶质细胞可能污染NEP...

小脑由多个细胞层，每层含有不同的细胞类型。在开发过程中，东瀛包含增殖颗粒神经元前体（国民生产总值），而分子层和浦肯野层包含伯格曼胶质细胞和浦肯野神经元，分别。小脑深处位于脑白质，其中包含神经干细胞（NSCs）和少突胶质细胞1。 音猬蛋白（Shh的）在调节小脑发育的关键作用，特别是，它促进的GNP的增殖通过与其受体结合补丁（PTC），对Shh信号2-4的负调节物。的Shh信号传导的异常活化产生髓母细胞瘤（MB），在孩子5,6中最常见的恶性脑肿瘤。 PTC突变MB的转基因小鼠模型是MB的学习功能强大的工具，开发肿瘤类似人类MB 7,8。使用这些小鼠，它被发现的GNP是起源于刺猬型MB 7的细胞。此外，除的GNP，我们最近发现的显影小脑也可以产生MB的EG​​L内的神经元祖细胞的独特群体。这些细胞表达高水平的VI型中间丝蛋白，巢蛋白，并且被称为网络设备供应商9。的NEP位于EGL的深部内并仅在新生儿小脑发育存在短暂。他们致力于颗粒细胞系作为国民生产总值却是不同的，因为它们是静态和不表达MATH1，一个完善的标志传统国民生产总值10。此外，网络设备供应商产生MB比更有效地活化Shh信号通路9，这使得它们的新型原点为MB的肿瘤发生后的GNP。 我们以前的显微切割技术分离出小脑东瀛非符合资格人士在4（P4）日龄这里说明9。通过组织直接镜检可视显微允许小脑东瀛的具体解剖。这是必要的，因为巢是由神经干细胞在小脑白质和伯格曼胶质细胞中的分子层1,7,11,12也表示，这是至关重要的，这些细胞不被包括在内，因为他们混淆分析。细胞显微组织分离可立即用于分子分析，或者它们可以被培养用于进一步的应用。 为了有助于具体microdissecting的EGL和进一步纯化的NEP，MATH1-GFP（绿色荧光蛋白）的小鼠进行杂交以巢蛋白-CFP（青色荧光蛋白）的小鼠。转录因子MATH1通过的GNP和GFP表达特异性表达是在EGL 9,13清晰可见。 巢蛋白-CFP小鼠表达CFP的核形式，并且可以很容易地可视化的分子层9,14。总之，GFP和CFP的表达创造边界东瀛的显微切割（ 见图1）。 CFP-阳性的NEP，然后通过FACS分离，继解剖EGLS的酶解。 目前，使用最多的方法也从东瀛分离细胞是整个小脑组织15,16 Percoll密度梯度离心法。这个方法，但是，不能完全排除从分子层和白质巢蛋白+细胞群中，因此不能被用于网络设备供应商的研究。激光捕获显微切割，其使用激光能量的传递，以除去所关注的细胞，是一种用于多相组织17,18内分离特定的细胞，但捕获的细胞仅可用于DNA，RNA和蛋白质的回收和不是另一种方法能够进行培养。 该协议提供了一种专门隔离东瀛非符合资格人士的活着，纯粹的人口。这种技术也可以应用于具有任何可识别的解剖结构或荧光标记细胞/区域不同的组织类型。因此，将这一新颖显微切割技术进入细胞的分离协议的主要优点是，细胞可以从特定组织区域进行分离，以消除污染邻近的组织和细胞可以立即被收集用于分析或培养的其他应用程序。

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			<td style="width:167px;">Comments/Description</td>
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			<td height="21" style="height:21px;width:215px;">Neurobasal media</td>
			<td style="width:71px;">Gibco</td>
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			<td height="21" style="height:21px;width:215px;">PDL</td>
			<td style="width:71px;">Millipore</td>
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			<td height="42" style="height:42px;width:215px;">ultra pure low melting temperature agarose</td>
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isolation of distinct cell populations from the developing cerebellum by microdissection

显微切割是一种新技术，可以分离组织的特定区域，消除污染邻近地区的细胞来源。在这项研究是第一个使用这种方法巢蛋白表达祖商（NEP），细胞在小脑外部生发层（EGL）的新发现的种群。在组合使用显微切割用荧光激活细胞分选（FACS），棉结的纯群体是从在EGL常规颗粒神经元前体和从其它污染巢蛋白表达细胞在小脑分开收集。未经显微切割，棉结的功能分析将不会有可能与可用的电流的方法，如Percoll密度梯度离心法和激光捕获显微解剖。该技术还可以应用于用于与包含任一可识别的区域或荧光标记细胞的各种组织中的使用。最重要的是，这主要microdissectio优势Ñ​​技术是分离的细胞是活的，并且可以被培养了进一步的实验，这是目前不可能与其他所描述的方法。

如图1A所示 ，小脑切片从P4 MATH1-GFP / 巢蛋白-CFP小鼠制备的，其中，所述小脑EGL主要是由绿色荧光蛋白表达的GNP和分子层中富集由CFP-阳性的神经胶质细胞。 （; 图1A中沿虚线）以分离的NEP位于EGL的深部的，切片的EGL和分子层之间的微切割。剖分EGLS（ 图1B）然后收集用酶解随后通过FACS。 正如预期的那样，细胞在解剖东瀛的大?...

Watch this Scientific Journal Video about Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection at JoVE.com

Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

从开发小脑显微切割分离的不同的细胞群

巢蛋白表达祖细胞是在发展中国家小脑的神经元祖细胞的新发现的种群。用在这里用荧光激活细胞分选组合呈现的显微切割技术中，该细胞群可与任何其他小脑区域污染被纯化和可以培养用于进一步研究。

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. ...

Research

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Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

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A Stainless Protocol for High Quality RNA Isolation from Laser Capture Microdissected Purkinje Cells in the Human Post-Mortem Cerebellum

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Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or ...