Name: isolation and culture of mouse cortical astrocytes
Uploaded: 2013-01-19T14:00:00.000+00:00
Duration: 11 min 25 s
Description: Watch this Scientific Journal Video about Isolation and Culture of Mouse Cortical Astrocytes at JoVE.com

SS，联邦教育与研究部（BMBF 01 EO 0803），KB和欧盟委员会FP7格兰特PIRG08-GA-2010-276989，NEUREX，德国研究基金会资助SCHA 1442 /的Fazit基金会研究生奖学金的支持下3-1 CS有没有冲突的财务权益。

1。混合皮层细胞的分离和电镀混合皮层星形胶质细胞培养细胞分离，可以使用P1到P4鼠标幼崽。为了实现适当的星形胶质细胞的密度，它是必要的使用4鼠标小狗皮质每T75组织培养烧瓶中。因此，以下协议中的卷是计算一个单元使用4次鼠标幼崽的准备。 <ol><li>开始解剖过程之前，prewarm 30毫升的星形胶质细胞的培养基（DMEM，高葡萄糖+ 10％热灭活的牛胎儿血清+ 1％青霉素/链霉素;见表）至37℃。涂布一T75烧瓶中，用20毫升的聚-D-赖氨酸（PDL），在1小时，在细胞培养级的水的浓度为50微克/毫升，在37℃下在CO 2培养箱中。 </li><li>对于解剖过程中，准备所有必要的试剂和材料。你将需要：手术剪，抚平细小的镊子，平头镊子，纸巾，垃圾袋，70％乙醇D 2解剖菜（3.5厘米直径）冰填充用2 ml HBSS。 </li><li>轻轻握住，并用70％乙醇的小狗鼠标喷的头部和颈部。被牺牲的动物通过斩首使用的剪刀。 </li><li>执行正中切口，后到前，，沿头皮透露的头骨。 </li><li>切头盖骨仔细地从脖子到鼻子。两个额外的削减，以便进一步进入大脑进行：第一刀前部的嗅球，另一种劣质的小脑断开的头盖骨，颅底。 </li><li>颅翼片使用扁平喷嘴钳，轻轻翻转到侧和大脑被取出并置于第一解剖菜填充用HBSS。将菜上的冰和继续收集所有4个大脑。 </li><li>在立体显微镜下进行解剖过程的其余部分。首先，在嗅球和小脑使用精细除去解剖钳（ 图1B）。 </li><li>为了找回皮质，抓住后用细镊子的大脑，执行一个正中切口两半球，镊子插入第二组创建的树林和剥离的板状结构的皮质大脑。 </li><li>仔细解剖脑膜皮质半球拉用细镊子（ 图1D）。此步骤可避免污染最终的星形胶质细胞的培养由脑膜细胞和成纤维细胞。准备皮质半球转移到第二盘充满了HBSS，并返回到冰。所有4个皮质继续进行。 </li><li>最后，横切成小块，用锋利的刀片（约4至8倍）的皮质半球。 </li><li>在无菌条件下，转印的皮质片放入一个50毫升Falcon管中，并添加至总体积为22.5毫升的HBSS中。 </li><li>加入2.5毫升的2.5％的胰蛋白酶，混合孵育的组织在37℃下在水浴中30分钟。混合偶尔晃动每10分钟。 </li><li>离心5分钟，在300×g离心沉淀皮层组织片。 </li><li>仔细收集上清用倾析法。为了避免失去组织沉淀，您可以机械地保留使用吸管。解离成单细胞悬浮液，通过添加10毫升星形胶质细胞电镀介质和剧烈的移液使用10毫升塑料移液管，直到组织片解离成单细胞（20〜30次）的组织。调节音量使用星形胶质细胞电镀液20毫升。您可以证明的皮质组织的离解成单个细胞，用血细胞计数板计数。一个准备的4鼠标小狗的皮质产生10-15×10 6个分离的单个细胞。 </li><li>吸干PDL从T75培养瓶中，板的解离的单细胞悬浮液和孵育在37℃下在CO 2培养箱中。 </li></ol> 2。获取ENRiched星形胶质细胞文化<ol><li>改变介质的电镀混合皮层细胞和所有的3天之后的2天之后。 </li><li> 7〜8天，当星形胶质细胞的汇合和叠加的小胶质细胞上的星形胶质细胞层坐在暴露或已经脱离的星形胶质细胞层（ 图2）后，摇动的T75烧瓶中，在180rpm下30分钟以除去小胶质细胞上轨道摇床。弃上清含小神经胶质细胞或文化，如果你想和检查小胶质细胞，旋转和文化28,29板。 </li><li>添加20毫升新鲜星形胶质细胞的培养基中，并继续通过摇动烧瓶中，在240 rpm下6小时，以除去少突胶质细胞的前体细胞（OPC）。由于一些OPCs的不会完全从星形胶质细胞层分离，的继续用手剧烈振摇1分钟，为了防止OPC污染。弃上清或旋转板，如果你想培养OPCs的29。 </li><li>冲洗剩余的confluent的星形胶质细胞层两次，用PBS，吸出PBS，添加5毫升胰蛋白酶-EDTA和孵育在37℃下在CO 2培养箱中每5分钟检查支队的星形胶质细胞和执行支队的星形胶质细胞的击中烧瓶中对你的手的手掌（2-3倍）。 </li><li>后星形胶质细胞的分离培养瓶中，加入5毫升的星形胶质细胞的培养液中，自旋细胞在180×g离心5分钟，吸出上清液，加40毫升新鲜的星形胶质细胞的镀介质。一个T75细胞培养瓶中后的第一次细胞分裂产生约1×10 6个细胞。板细胞在CO 2培养箱中在37℃下在两个T75培养瓶中并孵育。更改的介质每2到3天。 </li><li> 12-14天，先拆后星形胶质细胞接种于合适的细胞浓度为24-48小时，然后再进行实验。一个T75细胞培养瓶中应该产生大约1.5-2×10 6个细胞后，第二次细胞分裂。 </li></ol>

<ol>
	<li>Nedergaard, M., Ransom, B., Goldman, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+roles+for+astrocytes:+redefining+the+functional+architecture+of+the+brain.">New roles for astrocytes: redefining the functional architecture of the brain.</a> Trends Neurosci. 26, 523-530 (2003).</li><li>Belanger, M., Allaman, I., Magistretti, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+energy+metabolism:+focus+on+astrocyte-neuron+metabolic+cooperation.">Brain energy metabolism: focus on astrocyte-neuron metabolic cooperation.</a> Cell Metab. 14, 724-738 (2011).</li><li>Allen, N. J., 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=Neuroscience:+Glia+-+more+than+just+brain+glue.">Neuroscience: Glia - more than just brain glue.</a> Nature. 457, 675-677 (2009).</li><li>Ballas, N., Lioy, D. T., Grunseich, C., Mandel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-cell+autonomous+influence+of+MeCP2-deficient+glia+on+neuronal+dendritic+morphology.">Non-cell autonomous influence of MeCP2-deficient glia on neuronal dendritic morphology.</a> Nat. Neurosci. 12, 311-317 (2009).</li><li>Jacobs, S., Nathwani, M., Doering, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fragile+X+astrocytes+induce+developmental+delays+in+dendrite+maturation+and+synaptic+protein+expression.">Fragile X astrocytes induce developmental delays in dendrite maturation and synaptic protein expression.</a> BMC Neurosci. 11, 132 (2010).</li><li>Kaneko, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+neurons+clear+the+path+of+astrocytic+processes+for+their+rapid+migration+in+the+adult+brain.">New neurons clear the path of astrocytic processes for their rapid migration in the adult brain.</a> Neuron. 67, 213-223 (2010).</li><li>Min, R., Nevian, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+signaling+controls+spike+timing-dependent+depression+at+neocortical+synapses.">Astrocyte signaling controls spike timing-dependent depression at neocortical synapses.</a> Nat. Neurosci. , (2012).</li><li>Eroglu, C., 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=Regulation+of+synaptic+connectivity+by+glia.">Regulation of synaptic connectivity by glia.</a> Nature. 468, 223-231 (2010).</li><li>Sasaki, T., Matsuki, N., Ikegaya, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action-potential+modulation+during+axonal+conduction.">Action-potential modulation during axonal conduction.</a> Science. 331, 599-601 (2011).</li><li>Bozoyan, L., Khlghatyan, J., Saghatelyan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+control+the+development+of+the+migration-promoting+vasculature+scaffold+in+the+postnatal+brain+via+VEGF+signaling.">Astrocytes control the development of the migration-promoting vasculature scaffold in the postnatal brain via VEGF signaling.</a> J. Neurosci. 32, 1687-1704 .</li><li>Alvarez, J. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hedgehog+pathway+promotes+blood-brain+barrier+integrity+and+CNS+immune+quiescence.">The Hedgehog pathway promotes blood-brain barrier integrity and CNS immune quiescence.</a> Science. 334, 1727-1731 (2011).</li><li>Abbott, N. J., Ronnback, L., Hansson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-endothelial+interactions+at+the+blood-brain+barrier.">Astrocyte-endothelial interactions at the blood-brain barrier.</a> Nat. Rev. Neurosci. 7, 41-53 (2006).</li><li>Tao-Cheng, J. H., Nagy, Z., Brightman, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tight+junctions+of+brain+endothelium+in+vitro+are+enhanced+by+astroglia.">Tight junctions of brain endothelium in vitro are enhanced by astroglia.</a> J. Neurosci. 7, 3293-3299 (1987).</li><li>Gordon, G. R., Choi, H. B., Rungta, R. L., Ellis-Davies, G. C., MacVicar, 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=Brain+metabolism+dictates+the+polarity+of+astrocyte+control+over+arterioles.">Brain metabolism dictates the polarity of astrocyte control over arterioles.</a> Nature. 456, 745-749 (2008).</li><li>Silver, 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=Regeneration+beyond+the+glial+scar.">Regeneration beyond the glial scar.</a> Nat. Rev. Neurosci. 5, 146-156 (2004).</li><li>Schachtrup, C., Moan, N. L. e., Passino, M. A., Akassoglou, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+stellate+cells+and+astrocytes:+Stars+of+scar+formation+and+tissue+repair.">Hepatic stellate cells and astrocytes: Stars of scar formation and tissue repair.</a> Cell Cycle. 10, 1764-1771 (2011).</li><li>Schachtrup, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrinogen+triggers+astrocyte+scar+formation+by+promoting+the+availability+of+active+TGF-beta+after+vascular+damage.">Fibrinogen triggers astrocyte scar formation by promoting the availability of active TGF-beta after vascular damage.</a> J. Neurosci. 30, 5843-5854 (2010).</li><li>Bushong, E. A., Martone, M. E., Jones, Y. Z., Ellisman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protoplasmic+astrocytes+in+CA1+stratum+radiatum+occupy+separate+anatomical+domains.">Protoplasmic astrocytes in CA1 stratum radiatum occupy separate anatomical domains.</a> J. Neurosci. 22, 183-192 (2002).</li><li>Ogata, K., Kosaka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+quantitative+analysis+of+astrocytes+in+the+mouse+hippocampus.">Structural and quantitative analysis of astrocytes in the mouse hippocampus.</a> Neuroscience. 113, 221-233 (2002).</li><li>Dabir, D. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+glutamate+transport+in+a+mouse+model+of+tau+pathology+in+astrocytes.">Impaired glutamate transport in a mouse model of tau pathology in astrocytes.</a> J. Neuroscience. 26, 644-654 (2006).</li><li>Wisniewski, H. M., Wegiel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+relationships+between+astrocytes+and+classical+plaque+components.">Spatial relationships between astrocytes and classical plaque components.</a> Neurobiol. Aging. 12, 593-600 (1991).</li><li>Shin, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+mutant+huntingtin+in+glial+cells+contributes+to+neuronal+excitotoxicity.">Expression of mutant huntingtin in glial cells contributes to neuronal excitotoxicity.</a> J. Cell Biol. 171, 1001-1012 (2005).</li><li>Wakabayashi, K., Hayashi, S., Yoshimoto, M., Kudo, H., Takahashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NACP/alpha-synuclein-positive+filamentous+inclusions+in+astrocytes+and+oligodendrocytes+of+Parkinson's+disease+brains.">NACP/alpha-synuclein-positive filamentous inclusions in astrocytes and oligodendrocytes of Parkinson's disease brains.</a> Acta Neuropathol. 99, 14-20 (2000).</li><li>Lioy, D. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+glia+in+the+progression+of+Rett's+syndrome.">A role for glia in the progression of Rett's syndrome.</a> Nature. 475, 497-500 (2011).</li><li>Quinlan, R. A., Brenner, M., Goldman, J. E., Messing, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFAP+and+its+role+in+Alexander+disease.">GFAP and its role in Alexander disease.</a> Exp. Cell Res. 313, 2077-2087 (2007).</li><li>Beck, K., Schachtrup, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+damage+in+the+central+nervous+system:+a+multifaceted+role+for+vascular-derived+TGF-beta.">Vascular damage in the central nervous system: a multifaceted role for vascular-derived TGF-beta.</a> Cell Tissue Res. 347, 187-201 (2012).</li><li>McCarthy, K. D., de Vellis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+separate+astroglial+and+oligodendroglial+cell+cultures+from+rat+cerebral+tissue.">Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue.</a> J. Cell Biol. 85, 890-902 (1980).</li><li>Siao, C. J., Tsirka, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+plasminogen+activator+mediates+microglial+activation+via+its+finger+domain+through+annexin+II.">Tissue plasminogen activator mediates microglial activation via its finger domain through annexin II.</a> J. Neurosci. 22, 3352-3358 (2002).</li><li>Armstrong, R. 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+characterization+of+immature+oligodendrocyte+lineage+cells.">Isolation and characterization of immature oligodendrocyte lineage cells.</a> Methods. 16, 282-292 (1998).</li><li>Cahoy, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transcriptome+database+for+astrocytes,+neurons,+and+oligodendrocytes:+a+new+resource+for+understanding+brain+development+and+function.">A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function.</a> J. Neurosci. 28, 264-278 (2008).</li><li>Anthony, T. E., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+folate+metabolic+enzyme+ALDH1L1+is+restricted+to+the+midline+of+the+early+CNS,+suggesting+a+role+in+human+neural+tube+defects.">The folate metabolic enzyme ALDH1L1 is restricted to the midline of the early CNS, suggesting a role in human neural tube defects.</a> J. Comp. Neurol. 500, 368-383 (2007).</li><li>Skoff, R. P., Knapp, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Division+of+astroblasts+and+oligodendroblasts+in+postnatal+rodent+brain:+evidence+for+separate+astrocyte+and+oligodendrocyte+lineages.">Division of astroblasts and oligodendroblasts in postnatal rodent brain: evidence for separate astrocyte and oligodendrocyte lineages.</a> Glia. 4, 165-174 (1991).</li><li>Molofsky, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+and+disease:+a+neurodevelopmental+perspective.">Astrocytes and disease: a neurodevelopmental perspective.</a> Genes Dev. 26, 891-907 (2012).</li><li>Zhang, Y., 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=Astrocyte+heterogeneity:+an+underappreciated+topic+in+neurobiology.">Astrocyte heterogeneity: an underappreciated topic in neurobiology.</a> Curr. Opin. Neurobiol. 20, 588-594 (2010).</li><li>Foo, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+method+for+the+purification+and+culture+of+rodent+astrocytes.">Development of a method for the purification and culture of rodent astrocytes.</a> Neuron. 71, 799-811 (2011).</li><li>Jungblut, M., et al. <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+characterization+of+living+primary+astroglial+cells+using+the+new+GLAST-specific+monoclonal+antibody+ACSA-1.">Isolation and characterization of living primary astroglial cells using the new GLAST-specific monoclonal antibody ACSA-1.</a> Glia. 60, 894-907 (2012).</li></ol>

没有利益冲突的声明。

这里介绍的方法是基于从啮齿动物新生儿的大脑，于1980年27麦卡锡和德Vellis的原先所描述的星形胶质细胞培养制备。从出生P1到P4小鼠大脑皮质星形胶质细胞的分离和培养的方法，这里介绍的是速度快，产量纯星形胶质细胞是高度可重复性。这种技术可以很容易地转移到隔离来自其他物种的星形胶质细胞，如从大鼠或猪和由大脑的其他区域，如脊髓。而由麦卡锡和deVellis的方法，从新生儿?...

星形胶质细胞是中枢神经系统（CNS）中的一个非常丰富的细胞类型。在小鼠和大鼠皮质星形胶质细胞对神经元的比例是1:3，而每个神经元有1.4星形胶质细胞在人脑皮层1。在最近几年急剧增加星形胶质细胞功能的兴趣。一个关键功能的星形胶质细胞是神经元2,3结构和代谢支持的作用。新发现的星形胶质细胞的作用涵盖了广泛的功能。这些措施包括4-6在开发过程中，引导轴突和若干神经母细胞的迁移功能的突触传递，突触强度和信息处理的神经回路7-9，形成血脑屏障（BBB）的角色，在10和完整性11-13调节脑血管音14。的另一大特点是他们的星形胶质细胞对损伤的反应。在病理条件astrocyt的上课成为反应性和进一步上调表达的中间丝胶质纤维酸性蛋白（GFAP）和抑制细胞外基质（ECM）蛋白15,16。划定的健康组织的损伤部位形成胶质瘢痕，主要由星形胶质细胞分泌的细胞外基质蛋白，硫酸软骨素蛋白多糖（CSPG）家庭星形胶质细胞反应性增生，抑制轴突再生的主要因素，中枢神经系统损伤后15日至17日 。 星形胶质细胞在胚胎发育晚期和产后早期生命起源于放射状胶质细胞（RG）。规范已发生后，星形胶质细胞，星形胶质细胞的前体迁移到它们的最终位置，在那里他们开始终末分化过程中， 在体内 ，星形胶质细胞似乎是成熟的出生后三至四个星期，如所指示的典型形态18,19。 RG细胞亚群转化为脑室下区星形胶质细胞（B型细胞）。乙超视距，RG型和B细胞的功能在开发过程中的成人星形胶质细胞的神经干细胞（NS​​Cs）。喜欢的星形胶质细胞，RG和B型细胞也表达星形胶质细胞特异性的谷氨酸转运体（GLAST），脑脂质结合的蛋白（BLBP）的，和GFAP，表明这些标记可以不专门用于专门标记成人星形胶质细胞。相反成人的实质星形胶质细胞，没有分裂的健康的大脑，RG和B型细胞表现出干细胞的潜力，如自我更新的能力。星形胶质细胞失调有牵连的许多病症，包括老年痴呆症的20,21，亨廷顿氏病22，帕金森氏病23，Rett综合征24日和亚历山大的疾病25。此外，星形胶质细胞反应所有侮辱的中枢神经系统，导致星形胶质细胞活化和星形胶质细胞胶质瘢痕的形成16,26。星形胶质细胞胶质瘢痕形成以下大脑TRAUMA或脊髓损伤被认为是主要的屏障，防止神经细胞再生的15。 可靠的方法来隔离和保持纯化的细胞群的发展一直是我们了解神经系统的关键。创业的工作由麦卡锡和Vellis使调查人员，准备近纯培养的星形胶质细胞从新生大鼠组织27。关于使用这种方法，在这里稍加修改，以隔离小鼠脑皮层星形胶质细胞星形胶质细胞生物学已学到很多东西。补充体内研究中 ，星形胶质细胞以及空调介质使用所描述的体外培养，是宝贵的工具，以进一步深入了解星形胶质细胞的功能。

<table border="1"><tbody><tr><td> 工作解决方案的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 终浓度 </td></tr><tr><td></td><td></td><td></td><td> 星形胶质细胞培养基 </td></tr><tr><td> DMEM，高糖</td><td> Life Technologies公司</td><td> 31966-021 </td><td></td></tr><tr><td> FBS，热灭活</td><td> Life Technologies公司</td><td> 10082-1​​47 </td><td>终浓度：10％ </td></tr><tr><td>青霉素 - 链霉素</td><td> Life Technologies公司</td><td> 15140-122 </td><td>最终浓度：1％ </td></tr><tr><td></td><td></td><td></td><td> 脑组织掏的解决方案estion </td></tr><tr><td> HBSS </td><td> Life Technologies公司</td><td> 14170-088 </td><td></td></tr><tr><td> 2.5％胰蛋白酶</td><td> Life Technologies公司</td><td> 15090-046 </td><td>终浓度为0.25％ </td></tr><tr><td></td><td></td><td></td><td> 其他 </td></tr><tr><td> 70％（体积/体积）乙醇</td><td>罗斯</td><td> 9065.2 </td><td></td></tr><tr><td>聚-D-赖氨酸</td><td>密理博</td><td> A-003-E </td><td> 50微克/毫升</td></tr><tr><td>水</td><td> PAA </td><td> S15-012 </td><td>细胞培养级</td></tr><tr><td> PBS </td><td> PAA </td><td> H15-002 </td><td>细胞培养级</td></tr><tr><td> 0.05％的尝试PSIN-EDTA </td><td> Life Technologies公司</td><td> 25300-062 </td><td></td></tr><tr><td> 0.45微米的无菌过滤器</td><td>赛多利斯</td><td> 16555 </td><td></td></tr><tr><td> 3.5厘米培养皿</td><td> BD猎鹰</td><td> 353001 </td><td></td></tr><tr><td> 15毫升的Falcon管</td><td> BD猎鹰</td><td> 352096 </td><td></td></tr><tr><td> 50毫升Falcon管</td><td> BD猎鹰</td><td> 352070 </td><td></td></tr><tr><td> 75平方厘米的组织培养瓶中</td><td> BD猎鹰</td><td> 353136 </td><td></td></tr><tr><td>钳，细</td><td>杜蒙</td><td> 2-1032，2-1033 </td><td> ＃3C＃5 </td></tr><tr><td>钳，平头</td><td> KLS马丁</td><td> 12-120-11 </td><td></td></tr><tr><td> 13厘米的手术剪刀</td><td>蛇牌</td><td> BC-140-R </td><td></td></tr><tr><td>体视显微镜</td><td>徕卡</td><td></td><td> MZ7.5 </td></tr><tr><td>体视显微镜+相机</td><td>徕卡</td><td></td><td> MZ16F; DFC320 </td></tr><tr><td>显微镜+相机</td><td>蔡司，佳能</td><td></td><td>的普里莫垂直的PowerShot A650 IS </td></tr><tr><td>离心分离</td><td> Eppendorf公司</td><td> 5805000.017 </td><td> Centrifuge5804R </td></tr><tr><td>轨道振动筛</td><td> Thermo Scientific的</td><td> SHKE 4450-1CE </td><td> MAXQ 4450 </td></tr><tr><td>水浴</td><td> JULABO </td><td></td><td>小号W20 37°C; </td></tr></tbody></table>

isolation and culture of mouse cortical astrocytes

星形胶质细胞是哺乳动物大脑中的细胞类型丰富，但仍有许多工作有待了解它们的分子和功能特性。星形胶质细胞在体外培养系统可以用来详细研究这些神经胶质细胞的生物学功能。此视频协议显示了如何获得纯鼠标幼崽的混合皮层细胞的分离和培养的星形胶质细胞。该方法是基于上没有存活的神经元，星形胶质细胞，少突胶质细胞，小胶质细胞，三个主要的神经胶质细胞种群的中枢神经系统的，在培养和分离。在第一天的文化代表图像显示的混合细胞群的存在，并表示时间点，当星形胶质细胞的汇合和小胶质细胞和少突胶质细胞应分开。此外，我们表现出纯度和完善培养的星形胶质细胞，用免疫细胞化学染色星形胶质细胞形态和新近被描述的星形胶质细胞的标记。这种文化系统可以很容易地获得纯星形胶质细胞和星形胶质细胞条件培养液对星形胶质细胞生物学研究的各个方面。

当完整的小鼠脑组织（ 图1A），小脑和嗅球必须要除去（ 图1B）的隔离。小心地取出（ 图1E）的小鼠脑干个别皮层（ 图1D）（图1C）和脑膜皮层剥离。脑膜的是明显的由脑膜动脉系统的和不完整的去除污染由脑膜细胞和成纤维细胞，星形胶质细胞的最终文化结果。 电镀混合皮层细胞悬浮液后，某些星形胶质细胞已经附加到培?...

Watch this Scientific Journal Video about Isolation and Culture of Mouse Cortical Astrocytes at JoVE.com

Isolation and Culture of Mouse Cortical Astrocytes

星形胶质细胞被确认为是正常的大脑发育和功能，以及中枢神经系统的修复是必不可少的基本​​生物过程的多功能细胞参与。在这里，我们提出了一个快速的过程，以获得纯净的小鼠星形胶质细胞培养研究本类主要的中枢神经系统的细胞生物学。

小鼠脑皮层星形胶质细胞的分离和培养

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

isolation-and-culture-of-mouse-cortical-astrocytes

Research

JoVE Journal

Neuroscience

91.4K 次观看.  University of Freiburg. 星形胶质细胞被确认为是正常的大脑发育和功能，以及中枢神经系统的修复是必不可少的基本​​生物过程的多功能细胞参与。在这里，我们提出了一个快速的过程，以获得纯净的小鼠星形胶质细胞培养研究本类主要的中枢神经系统的细胞生物学。

视频: Isolation and Culture of Mouse Cortical Astrocytes

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

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

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 ...

科研

免疫与感染

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

建模星形细胞瘤发病机制<em&gt;在体外</em&gt;和<em&gt;体内</em&gt;使用星形胶质细胞或条件，遗传工程小鼠神经干细胞

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

神经科学

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies indicate that the severity of brain injury appear to be sex dependent, and that the male neonates are more susceptible to the effects of HI-related brain injury, resulting in more severe neurological outcomes as compared to females with comparable brain injuries. The development of reliable methods to isolate and maintain highly enriched populations of sexed hippocampal astrocytes is essential to understand the cellular basis of sex differences in the pathological consequences of neonatal HI. In this study, we describe a method for creating sex specific hippocampal astrocyte cultures that are subjected to a model of in-vitro ischemia, oxygen-glucose deprivation, followed by reoxygenation. Subsequent reactive astrogliosis was examined by immunostaining for the Glial Fibrillary Acidic Protein (GFAP) and S100B. This method provides a useful tool to study the role of male and female hippocampal astrocytes following neonatal HI, separately.

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Astrocytes are one of the most important key players in the central nervous system (CNS). Here, we are reporting a practical method of sexed hippocampal astrocyte culture protocol in order to study the mechanisms underlying the astrocyte function in male and female neonate pups after in-vitro ischemia.

性别差异在小鼠海马星形胶质细胞<em&gt;在体外</em&gt;缺血

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

间接神经元的星形胶质细胞共培养试验:一个<em&gt;体外</em&gt;设置 - 为神经元的神经胶质细胞相互作用的详细调查

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in brain homeostasis, astrocytes supply neurons with vital metabolites and buffer extracellular water, ions, and glutamate. An integral component of the &#8220;tri-partite&#8221; synapse, astrocytes are also critical in the formation, pruning, maintenance, and modulation of synapses. To enable these highly interactive functions, astrocytes communicate among themselves and with other glial cells, neurons, the brain vasculature, and the extracellular environment through a multitude of specialized membrane proteins that include cell adhesion molecules, aquaporins, ion channels, neurotransmitter transporters, and gap junction molecules. To support this dynamic flux, astrocytes, like neurons, rely on tightly coordinated and efficient intracellular transport. Unlike neurons,&#160;where intracellular trafficking has been extensively delineated, microtubule-based transport in astrocytes has been less studied. Nonetheless, exo- and endocytic trafficking of cell membrane proteins and intracellular organelle transport orchestrates astrocytes&#8217; normal biology, and these processes are often affected in disease or in response to injury. Here we present a straightforward protocol to culture high quality murine astrocytes, to fluorescently label astrocytic proteins and organelles of interest, and to record their intracellular transport dynamics using time-lapse confocal microscopy. We also demonstrate how to extract and quantify relevant transport parameters from the acquired movies using available image analysis software (i.e., ImageJ/FIJI) plugins.

Here we describe an in vitro live-imaging method to visualize intracellular transport of organelles and trafficking of plasma membrane proteins in murine astrocytes. This protocol also presents an image analysis methodology to determine cargo transport itineraries and kinetics.

可视化和分析星形细胞中细胞内器官和其他货物的细胞内传输

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in ...

Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through multipotent intermediates. This technique not only holds great promises in the field of disease modeling, as it allows to convert, for example, fibroblasts for patients suffering neurodegenerative diseases into neurons, but also represents a promising alternative for cell-based replacement therapies. In this context, a major scientific breakthrough was the demonstration that differentiated non-neural cells within the central nervous system, such as astrocytes, could be converted into functional neurons in vitro. Since then, in vitro direct reprogramming of astrocytes into neurons has provided substantial insights into the molecular mechanisms underlying forced identity conversion and the hurdles that prevent efficient reprogramming. However, results from in vitro&#160;experiments performed in different labs are difficult to compare due to differences in the methods used to isolate, culture, and reprogram astrocytes. Here, we describe a detailed protocol to reliably isolate and culture astrocytes with high purity from different regions of the central nervous system of mice at postnatal ages via magnetic cell sorting. Furthermore, we provide protocols to reprogram cultured astrocytes into neurons via viral transduction or DNA transfection. This streamlined and standardized protocol can be used to investigate the molecular mechanisms underlying cell identity maintenance, the establishment of a new neuronal identity, as well as the generation of specific neuronal subtypes and their functional properties.

Here we describe a detailed protocol to generate highly enriched cultures of astrocytes derived from different regions of the central nervous system of postnatal mice and their direct conversion into functional neurons by the forced expression of transcription factors.

小鼠星形胶质细胞的分离和直接神经元重编程

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through ...

从成年小鼠脊髓中高效提取星形胶质细胞和小胶质细胞

Isolation of Pure Astrocytes and Microglia from the Adult Mouse Spinal Cord For In Vitro Assays and Transcriptomic Studies

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic cells exhibit rapid responses to environmental changes and display significant heterogeneity in terms of morphology, transcriptional profiles, and functions. While our understanding of the functions of glial cells in health and disease has advanced substantially, there remains a need for in vitro, cell-specific analyses conducted in the context of insults or injuries to comprehensively characterize distinct cell populations. Isolating cells from the adult mouse offers several advantages over cell lines or neonatal animals, as it allows for the analysis of cells under pathological conditions and at specific time points. Furthermore, focusing on spinal cord-specific isolation, excluding brain involvement, enables research into spinal cord pathologies, including experimental autoimmune encephalomyelitis, spinal cord injury, and amyotrophic lateral sclerosis. This protocol presents an efficient method for isolating astrocytes and microglia from the adult mouse spinal cord, facilitating immediate or future analysis with potential applications in functional, molecular, or proteomic downstream studies.

作者聚焦:从成年小鼠脊髓中分离神经胶质细胞 

This protocol outlines the isolation of purified astrocytes and microglia from the adult mouse spinal cord, facilitating subsequent applications such as RNA analysis and cell culture. It includes detailed cell dissociation methods and procedures designed to enhance both the quality and yield of isolated cells.

从成年小鼠脊髓中分离纯星形胶质细胞和小胶质细胞，用于 体外 测定和转录组学研究

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic ...

Isolation of Microglia from the Cortex of an Adult Mouse Brain

Source: Zhou, L., et al., <a href="https://app.jove.com/v/60347">Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing.</a> J. Vis. Exp. (2019)
The video demonstrates a technique for isolating microglia from the cortex of an adult mouse brain. First, the cortex undergoes segmentation and homogenization to remove neurons and other glial cells, preserving microglia. Following that, the cell suspension is exposed to anti-myelin magnetic microbeads and a magnetic field, effectively separating microglia and myeloid cells from myelin debris and cells containing myelin.

从成年小鼠大脑的皮层中分离小胶质细胞

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Mechanical Tissue Dissociation
...

小鼠脑皮层星形胶质细胞的分离和培养

摘要

探索更多视频

此视频中的章节

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

建模星形细胞瘤发病机制

性别差异在小鼠海马星形胶质细胞

间接神经元的星形胶质细胞共培养试验:一个

可视化和分析星形细胞中细胞内器官和其他货物的细胞内传输

小鼠星形胶质细胞的分离和直接神经元重编程

从成年小鼠脊髓中分离纯星形胶质细胞和小胶质细胞，用于体外测定和转录组学研究

从成年小鼠脊髓中分离纯星形胶质细胞和小胶质细胞，用于体外测定和转录组学研究

从成年小鼠脊髓中分离纯星形胶质细胞和小胶质细胞，用于体外测定和转录组学研究

从成年小鼠大脑的皮层中分离小胶质细胞

小鼠脑皮层星形胶质细胞的分离和培养

摘要

探索更多视频

此视频中的章节

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

建模星形细胞瘤发病机制

性别差异在小鼠海马星形胶质细胞

间接神经元的星形胶质细胞共培养试验:一个

可视化和分析星形细胞中细胞内器官和其他货物的细胞内传输

小鼠星形胶质细胞的分离和直接神经元重编程

从成年小鼠脊髓中分离纯星形胶质细胞和小胶质细胞，用于 体外 测定和转录组学研究

从成年小鼠脊髓中分离纯星形胶质细胞和小胶质细胞，用于 体外 测定和转录组学研究

从成年小鼠脊髓中分离纯星形胶质细胞和小胶质细胞，用于 体外 测定和转录组学研究

从成年小鼠大脑的皮层中分离小胶质细胞

从成年小鼠脊髓中分离纯星形胶质细胞和小胶质细胞，用于体外测定和转录组学研究

从成年小鼠脊髓中分离纯星形胶质细胞和小胶质细胞，用于体外测定和转录组学研究

从成年小鼠脊髓中分离纯星形胶质细胞和小胶质细胞，用于体外测定和转录组学研究