Name: isolation and culture of rat embryonic neural cells: a quick protocol
Uploaded: 2012-05-24T12:00:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol at JoVE.com

我们感谢社论援助乔娜埃利斯。项目描述奖号码R01MH079751（主持人：F.佩鲁齐）从国家心理健康研究所的支持。内容完全是作者的责任，并不代表国家精神卫生研究所或国立卫生研究院的官方意见。

1。聚-D-赖氨酸（PDL）的制备<ol><li>加入5毫升无菌DDH 2至5毫克的客运专线，获得1毫克/毫升的原液。 </li><li>混合原液吹打几次。 </li><li>立即使用或储存聚-D-赖氨酸的解决方案，在2-8°C。 </li></ol> 2。聚-D-赖氨酸（PDL）：涂层塑料细胞培养皿<ol><li> PDL原液稀释用无菌DDH 2 O的终浓度为10微克/毫升。 </li><li>吸取足够的解决方案，覆盖养殖水面面积为60毫米的菜（3毫?...

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	<li>Aprea, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tubulin-mediated+binding+of+human+immunodeficiency+virus-1+Tat+to+the+cytoskeleton+causes+proteasomal-dependent+degradation+of+microtubule-associated+protein+2+and+neuronal+damage.">Tubulin-mediated binding of human immunodeficiency virus-1 Tat to the cytoskeleton causes proteasomal-dependent degradation of microtubule-associated protein 2 and neuronal damage.</a> J. Neurosci. 26, 4054-4062 (2006).</li><li>Kivell, B. M., McDonald, F. J., Miller, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-free+culture+of+rat+post-natal+and+fetal+brainstem+neurons.">Serum-free culture of rat post-natal and fetal brainstem neurons.</a> Brain Res. Dev. Brain Res. 120, 199-210 (2000).</li><li>Kivell, B. M., McDonald, F. J., Miller, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+serum-free+culture+of+late+fetal+and+early+postnatal+rat+brainstem+neurons.">Method for serum-free culture of late fetal and early postnatal rat brainstem neurons.</a> Brain Res. Brain Res. Protoc. 6, 91-99 (2001).</li><li>Brewer, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-free+B27/neurobasal+medium+supports+differentiated+growth+of+neurons+from+the+striatum,+substantia+nigra,+septum,+cerebral+cortex,+cerebellum,+and+dentate+gyrus.">Serum-free B27/neurobasal medium supports differentiated growth of neurons from the striatum, substantia nigra, septum, cerebral cortex, cerebellum, and dentate gyrus.</a> J. Neurosci. Res. 42, 674-683 (1995).</li><li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing nerve cells. , (1998).</li><li>Eletto, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+SNAP25+expression+by+HIV-1+Tat+involves+the+activity+of+mir-128a.">Inhibition of SNAP25 expression by HIV-1 Tat involves the activity of mir-128a.</a> J. Cell Physiol. 216, 764-770 (2008).</li><li>Gualco, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IGF-IR-dependent+expression+of+Survivin+is+required+for+T-antigen-mediated+protection+from+apoptosis+and+proliferation+of+neural+progenitors.">IGF-IR-dependent expression of Survivin is required for T-antigen-mediated protection from apoptosis and proliferation of neural progenitors.</a> Cell Death Differ. 17, 439-451 (2010).</li><li>Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+and+differentiation+of+adult+neuronal+progenitor+cells+transplanted+to+the+adult+brain.">Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain.</a> Proc. Natl. Acad. Sci. U.S.A. 92, 11879-11883 (1995).</li><li>Keyser, D. O., Pellmar, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+transmission+in+the+hippocampus:+critical+role+for+glial+cells.">Synaptic transmission in the hippocampus: critical role for glial cells.</a> Glia. 10, 237-243 (1994).</li><li>Pfrieger, F. W., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+efficacy+enhanced+by+glial+cells+in+vitro.">Synaptic efficacy enhanced by glial cells in vitro.</a> Science. 277, 1684-1687 (1997).</li></ol>

大鼠海马和皮层神经元的解剖和文化在这里介绍的方法，允许使用在化学上定义的介质（ 图3）增长了近纯神经细胞进行实验。虽然近纯无血清培养基中培养神经元的协议已先前所述的2,3,4，也有我们的方法中作出了重要的变化。不同于传统的协议（即银行家等）5，我们已取代TrypLE快速，更温和的分解酶胰蛋白酶。我们也省略了两个步骤，这可能会影响神经细胞的完?...

<table border="1"><tbody><tr><td> 试剂 </td><td> 浓度 </td></tr><tr><td>的Neurobasal </td><td> 98％ </td></tr><tr><td> B27的</td><td> 2％ </td></tr><tr><td> glutamax </td><td> 0.5毫米</td></tr></tbody></table> 表一Neurobasal/B27完全培养基 。 <table border="1"><tbody><tr><td> 试剂 </td><td> 浓度 </td></tr><tr><td>葡萄糖</td><td> 16毫米</td></tr><tr><td>蔗糖</td><td> 22毫米</td></tr><tr><td>肝素钠</td><td> 10毫米</td></tr><tr><td>氯化钠</td><td> 160毫米</td></tr><tr><td>氯化钾</td><td> 5毫米</td></tr><tr><td>的Na 2 HPO 4 </td><td> 1毫米</td></tr><tr><td> KH 2 PO 2 </td><td> 0.22毫米</td></tr><tr><td>庆大霉素</td><td> 50微克/毫升</td></TR&gt; <tr><td> fungizone </td><td> 250毫微克/毫升</td></tr><tr><td> pH值</td><td> 7.4 </td></tr><tr><td>渗透压</td><td> 320-330 mOsm </td></tr></tbody></table> 表二。夹层介质 。 <table border="1"><tbody><tr><td> 试剂 </td><td> 量（μl） </td></tr><tr><td> Neurobasal/B27完整的介质</td><td> 240 </td></tr><tr><td>台盼蓝染色0.4％ </td><td> 250 </td></tr><tr><td>总</td><td> 490 </td></tr></tbody></table> 表三。 50X计算解决方案 。 <table border="1"><tbody><tr><td> 试剂 </td><td> 公司 </td><td> 猫。数 </td></tr><tr><td> Hibernate的é </td><td> brainbits </td><td> 767171 </td></tr><tr><td>的Neurobasal </td><td> Gibco公司，Invitrogen公司</td><td> 21103-049 </td></tr><tr><td> B27的</td><td> Gibco公司，Invitrogen公司</td><td> 17504-044 </td></tr><tr><td> fungizone </td><td> Gibco公司，Invitrogen公司</td><td> 15290-018 </td></tr><tr><td>硫酸庆大霉素</td><td> Sigma Aldrich公司</td><td> G1264 </td></tr><tr><td> glutamax 200毫米</td><td> Gibco公司，Invitrogen公司</td><td> 35050 </td></tr><tr><td> TrypLE快递W / O酚红</td><td> Gibco公司，Invitrogen公司</td><td> 12604 </td></tr><tr><td>胞嘧啶β-D-阿糖盐酸盐</td><td> Sigma Aldrich公司</td><td> C6645 </td></tr><tr><td>聚-D-赖氨酸</td><td> Sigma Aldrich公司</td><td> P6407 </td></tr><tr><td>层粘连蛋白1毫克/毫升</td><td> Millipore公司</td><td> CC095 </td></tr><tr><td>肝素钠</td><td> Sigma Aldrich公司</td><td> H3375 </td></tr><tr><td>台盼蓝染色0.4％ </td><td> Gibco公司，Invitrogen公司</td><td> 15250 </td></tr></tbody></表&gt; 表四。特定的试剂 。 <table border="1"><tbody><tr><td> 设备 </td><td> 公司 </td><td> 猫。数 </td></tr><tr><td>立体显微镜</td><td>奥林巴斯</td><td> SZ61 </td></tr><tr><td>大钳</td><td>福斯特</td><td> 11022-14 </td></tr><tr><td>细尖镊子</td><td>莫里亚</td><td> MC40B </td></tr><tr><td>微精尖镊子</td><td>莫里亚</td><td> MC31 </td></tr><tr><td>剃刀般锋利的剪刀</td><td> roboz </td><td> RS-6820 </td></tr><tr><td>微解剖剪刀</td><td>福斯特</td><td> 91460-11 </td></tr><tr><td>微解剖弯剪刀</td><td>福斯特</td><td> 14067-11 </td></tr><tr><td>玻璃2玻片</td><td>实验室康</td><td> 154461 </td></tr><tr><td> 60毫米菜</td&gt; <td>屋宇署猎鹰</td><td> 353002 </td></tr><tr><td> 100毫米菜</td><td>康宁</td><td> 430167 </td></tr><tr><td> 15毫升管</td><td>屋宇署猎鹰</td><td> 352099 </td></tr><tr><td> 1.5毫升冷冻管瓶</td><td> NUNC </td><td> 375353 </td></tr></tbody></table> 表五，特殊设备 。

isolation and culture of rat embryonic neural cells: a quick protocol

我们描述了一个快速的方法分离和培养E15-17大鼠胚胎海马或皮层神经元。程序可以成功地应用于老鼠和人类的初级神经元和神经祖细胞的分离。分离神经元无血清培养基均保持在几个星期。这些文化可用于核转染，免疫细胞化学，核酸制备，以及电。年老的神经细胞，也可以慢病毒转导效率，有效地减少，磷酸钙或脂质的方法，如脂质体转染。

Watch this Scientific Journal Video about Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol at JoVE.com

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

我们描述了一个快速的方法分离和培养的海马和皮层神经细胞从老鼠胚胎。该协议允许在几乎纯神经细胞需要我们进行实验。

大鼠胚胎神经细胞的分离和培养：一个快速议定书“

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Research

JoVE Journal

Neuroscience

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

成年鼠神经干细胞的分离和扩大使用神经干含量

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

建立小鼠胚胎神经干细胞培养神经球含量

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

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). ...

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

从人类毛囊神经嵴干细胞的分离和培养的

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

基于凝集素的分离和培养的小鼠胚胎运动神经元

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

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 ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

解剖，文化与分析<em&gt;非洲爪蟾</em&gt;胚胎视网膜组织

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

从胚胎前脑内皮细胞的分离和培养

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

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 ...