Name: the indirect neuron-astrocyte coculture assay: an in vitro set-up for the detailed investigation of neuron-glia interactions
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Description: Watch this Scientific Journal Video about The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions at JoVE.com

The present work was supported by the German research foundation (Deutsche Forschungsgemeinschaft DFG: GRK 736, Fa 159/22-1; the research school of the Ruhr University Bochum (GSC98/1) and the priority program SSP 1172 "Glia and Synapse", Fa 159/11-1,2,3).

小鼠实验是按照德国法律和德国社会为畜牧业的神经科学的指导方针。在波鸿鲁尔大学的动物保护与利用委员会授予相应的许可证。 1.准备和皮层星形胶质细胞的培养注:进行下一个步骤，有准备的神经元前的星形胶质细胞的文化应该发展成融合单层之前完成这些步骤，该协议至少7天的。原发性星形胶质细胞从各地日龄（P），0-3的乳鼠获得的混合胶质细胞培养的。每T75烧瓶三大脑（6皮层）都可以使用。 <ol><li>通过用无菌条件下10％体积/体积马血清和0.1％体积/体积庆大霉素补充的Dulbecco改良的Eagle培养基（DMEM）制备星形细胞介质。储存在4℃的培养基中达2周。 </li><li>大衣3倍T75瓶无线第10微克/毫升聚-D-赖氨酸（PDL;在细胞培养级水中）在37℃下在6％的体积/体积的 CO 2的存在下至少1小时。涂布后，用磷酸盐缓冲盐水（PBS）洗烧瓶两次以除去未结合的阳离子，并添加9毫升星形细胞培养基。 </li><li>对于大脑的制备中，加入10毫升Hank氏平衡盐溶液（HBSS）为1x 10cm培养皿和1mL的HBSS至1x 15毫升管（无论将要使用的大脑的数量）。保持一双手术剪，2细镊子，并在过程中达到双目。 </li><li>快速斩首3幼仔的手术剪和收集元首在一个空的10cm平皿。 <ol><li>执行双目下的皮质的准备。使用两个细镊子，在中线删除从头骨的背部皮肤，从颈部，迈向眼睛的水平开始。此后，其血管，大脑颅骨下可见。 </li><li>在一双镊子的喙端固定头切开颅骨的中线，开始在后端。接着，夹断以暴露脑的右和头骨的左侧半部。 </li><li>关闭钳子的前端和脑下它腹侧的位置。解除脑出颅骨，并转移到HBSS填充10cm培养皿。与剩余的标本继续以同样的方式，直到所有的大脑中的菜收集。 </li></ol></li><li>收集大脑后，开始与皮质通过夹断使用一对像一把剪刀镊子的后脑的分离。一个钳子执行两半球之间的正中切口。仔细固定用钳子脑和切断脑和使用第二镊子嗅球与皮质半部到结束。 </li><li>修复一个半皮质与一个钳子和拆卸他们˚F剥离从半球脑膜ROM中的横向边缘，并使用第2钳子从皮质表面随后拉动它们。 </li><li>翻转皮质一半在其表面面向下来，对盘中;仔细剖析并删除月牙形海马使用一个钳子。转移单皮质成使用一个封闭的镊子进行个别皮质锥形15毫升管。 </li><li>收集所有皮质，过渡到无菌层流罩，并与筹备皮层组织的解离后开始。 </li><li>添加1毫升的DMEM到一个2毫升的反应管中，加入0.1％w / v的木瓜蛋白酶（30单位）。孵育在水浴悬浮液，在37℃直至澄清溶液开发（约3分钟）。添加0.24毫克/毫升的L-半胱氨酸和20微克/毫升DNA酶，轻轻摇晃。 注:对于消化，需要约1毫升酶液。 </li><li>过滤器（0.22微米孔径）将之前获得1毫升无菌溶液它的皮层组织。温育30分钟的管- 1小时，在37℃（ 即在水浴中）。 注意:步骤1.11 - 1.14是关键的，应在15分钟内完成。 </li><li>终止消化反应，加1毫升星形胶质细胞中，并用1毫升吸管仔细磨碎消化的组织。进行研制，轻轻移动吸移管柱塞，从而吸移和挤出皮层组织悬浮液中。 </li><li>一旦组织已被解离成单细胞悬浮液中，加入5毫升星形细胞介质和离心机在216×g下5分钟。 </li><li>从所得的沉淀小心吸出上清液并重新悬浮于1mL星形胶质细胞培养基中的细胞。 </li><li>细胞悬浮液加入到9毫升星形胶质细胞培养基（见1.2）补充T75瓶。 </li><li>孵育所述细胞在37℃下在6％的体积/体积的 CO 2的存在下孵化。 4 d后，执行第一次完全培养基变化（10毫升）中。此后，改变培养基，每2 - 3天。 </li><li> 7天后，检查细胞形成融合单层。如果没有，再等2 - 3天的文化完全融合直至达到。 注:星形胶质细胞显示大扁平细胞体，定位在烧瓶底部，不开发显着的细胞过程。他们从小相衬亮祖细胞和小胶质驻留在单层17的顶部有所不同。 </li><li>为了摆脱祖细胞，并获得纯星形胶质细胞文化，将T75瓶上轨道摇床，动摇文化O / N在250转。确保温度调节至37℃，并且将烧瓶中的过滤器是用实验室膜密封，以防止二氧化碳的蒸发。 </li><li>第二天，吸出培养基，并添加有20μM的胞嘧啶-1-β-D-阿拉伯呋喃糖苷（阿糖胞苷）补充10mL的新鲜培养基来eliminaTE残留分裂的细胞。 注:孵育阿糖胞苷2 -在37℃下在孵化3天，6％的CO 2，将导致一个纯星形胶质细胞培养。 </li><li>监视星形细胞培养的通过视觉检查在光学显微镜下的纯度。如果残留相衬亮祖细胞和小胶质细胞仍驻留在星形细胞单层17的顶部（见步骤1.16），重复步骤1.18和1.19。 注:汇合和纯星形胶质细胞单层可在继续下一步骤之前被保持在孵化器达14天（37℃，6％体积/体积的 CO 2）。一半的培养基改变每3天。不收集，冻结和解冻的星形胶质细胞，因为这些细胞会通过存储过程失去神经的支持性能。 </li></ol> 2.准备星形胶质细胞的间接共培养注:48 - 72小时准备神经元，星形胶质细胞转移到CEL前L-培养插入。通常，一个单一的T75烧瓶提供了足够数量的细胞，以支持与一种制备得到的神经元培养物。 <ol><li>根据需要于24孔板将尽可能多的插入。外套每个插入用10微克/毫升的PDL（溶于细胞培养级水中）下进行约1小时，在37℃，6％体积/体积的 CO 2。培养后，用PBS洗插入两次。在此期间，继续星形胶质细胞的准备。 注:填充有PBS中插入可以保持，直到星形胶质细胞悬浮液是准备使用。 </li><li>吸星形细胞培养基（与阿糖胞苷）中，用10毫升PBS洗涤培养一次，以确保任何剩余的血清被去除。 </li><li>加入3毫升0.05％胰蛋白酶-EDTA（乙二胺四乙酸）到烧瓶孵育胰蛋白酶将细胞在37℃，6％体积/体积的 CO 2约10分钟。 </li><li>控制通过目测用显微镜和facilita的细胞脱离通过敲击针对桌面烧瓶的侧TE上的细胞的分离。 注意:步骤2.5 - 2.8是关键的，并应15内完成 - 20分钟。 </li><li>继胰蛋白酶，轻轻重新悬浮在7mL的星形胶质细胞培养基中的细胞和细胞悬浮液转移到15毫升管中。离心机在216×g下5分钟。 </li><li>小心吸出上清液并重新悬浮于1mL星形胶质细胞培养基中的细胞沉淀。 </li><li>算使用计数室中的细胞。 </li><li>填补24孔板用500微升星形胶质细胞培养基的孔的底部和25,000个细胞在500μL的星形胶质细胞培养基转移到每个单独的插入物。孵育培养在37℃，6％体积/体积的 CO 2。 注:42后 - 72小时的培养将达到约100％汇合。在这一阶段，培养的纯度可以通过免疫细胞化学11进行验证。汇合培养物可维持到7天，直到继续到下一步。一半的培养基改变每3天。 </li></ol> 3.初级海马神经元的制备注:主小鼠海马神经元应从E15.5导出 - 定时孕鼠胚胎E16。 <ol><li>制备神经元培养基（MEM用10mM丙酮酸钠，0.1％w / v的卵清蛋白，2％体积/体积B27培养基补充剂，和0.1％体积/体积庆大霉素（无菌过滤）），并且制备培养基（HBSS用0.6％重量/ v葡萄糖和10mM的HEPES（4-（2-羟乙基）-1-哌嗪乙磺酸））。预先温暖和预平衡在过滤烧瓶神经元培养基中在37℃，6％体积/体积的 CO 2。 </li><li>放置玻璃盖玻片（12毫米直径）到24孔板的孔中（使用特殊的盖玻片，其缺口与细胞培养插入的使用）。 <ol><li>外套与15微克/毫升聚-DL-鸟氨酸在水（细胞培养级）在37℃下至少1小时。至少使用500微升每孔，以保证盖玻片完全覆盖。用PBS洗涤各孔两次。离开PBS中的孔，直到镀覆的细胞。确保孔不干燥。 </li></ol></li><li>解剖海马，准备3倍10厘米充满制备介质菜肴和1个2毫升管1毫升的准备中。准备好剪刀和2细镊子。 </li><li>通过颈脱位牺牲怀孕小鼠，消毒用70％V / V乙醇洗涤腹部，并用锋利的剪刀打开腹部。用剪刀仔细地切除子宫串珠和器官转移到1X 10cm皿充满了制备介质。 </li><li>接下来，请从子宫的胚胎。小心切开子宫，取出羊膜，而不使用2钳伤害胚胎。此后，斩首用剪刀的每个胚胎和磁头转移到与制备介质提供的第二10cm皿。 </li><li>与头的准备（类似prepar继续1.7） - 通货膨胀在1.4描述。 <ol><li>固定有一对镊子的头部和切割在颈部区域中的颅骨和皮肤靠近后脑，垂直于神经轴。使用第二对镊子解除两者头骨和覆盖皮肤和弯曲朝向头部的喙端软颅盖，从而露出大脑。 </li><li>为2钳子的帮助下，从颅腔分离大脑。要做到这一点，接近1钳子和脑启动在嗅球，并朝着移动后脑颅骨分离。收集再过10 cm培养皿充满制备介质的大脑。 </li></ol></li><li>如上对于星形细胞制备（步骤1.6）中描述从皮质部分解剖海马。从每个半球中取出海马，并收集他们在2毫升管中充满制备介质。 </li><li>在完成剥离之后，将管转移到无菌的层流工作台和diges吨，含木瓜蛋白酶1毫升消解液中的海马组织。如步骤1.9所述，准备消解液 - 1.10，但使用MEM代替DMEM。 注意:步骤3.9 - 3.12是关键的，应10内完成 - 15分钟。 </li><li>消化完成后，小心地撤回通过温和抽吸用移液器将消化溶液。 </li><li>依次添加，然后小心地吸每个洗涤周期1毫升新鲜培养基清洗海马神经元与介质3倍。不要离心细胞悬液。 </li><li>最后的洗涤步骤之后，请仔细磨碎在1毫升神经元中的组织。 </li><li>算使用计数室对细胞和板35,000个细胞在500μL的神经介质每一个24孔板（在3.2中提到的板）的单个孔。 注意:任选地，细胞等分试样可以用台盼蓝进行复染，以评估细胞制剂的活力。控制通过视觉检查接种密度离子使用显微镜（一般为90 - 每一个标准10X物镜的视场110细胞）。 </li><li>在孵化器处的神经元，在37℃，1小时6％体积/体积的 CO 2。 </li><li> （去籽与融合星形胶质细胞单层）从孵化器中孵化后，把准备好的插件（见步骤2.8）。通过抽吸星形胶质细胞中，用500微升新鲜的神经元中替换它交换媒介。 注意:应插入后2怀有星形胶质细胞融合单层- 3 DIV（天体外 ）。 </li><li>将带有星形胶质细胞刀片小心地插入包含神经元文化（3.13步），使用无菌镊子井。 </li><li>放置导致的间接神经元星形胶质细胞共培养放回培养箱中于37℃，6％体积/体积的 CO 2。 注意:在这些条件下，神经元可以在完全确定的培养基中培养长达4周。无介质的改变是必要的。对于长期培养物时，建议以填充ÈMPTY井用无菌水，以减少从24孔板的蒸发。 </li><li>进行体外细胞分析所需的培养后时段（ 例如，免疫细胞化学，PCR，Western印迹，电生理记录（膜片钳或者多电极阵列））7,15,18。 注:培养系统也适合于培养11急性和慢性药物治疗。 </li></ol>

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S., 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=Thrombospondins+are+astrocyte-secreted+proteins+that+promote+CNS+synaptogenesis.">Thrombospondins are astrocyte-secreted proteins that promote CNS synaptogenesis.</a> Cell. 120, 421-433 (2005).</li><li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+hippocampal+neurons.">Culturing hippocampal neurons.</a> Nat Protoc. 1, 2406-2415 (2006).</li><li>Geissler, 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=Primary+hippocampal+neurons,+which+lack+four+crucial+extracellular+matrix+molecules,+display+abnormalities+of+synaptic+structure+and+function+and+severe+deficits+in+perineuronal+net+formation.">Primary hippocampal neurons, which lack four crucial extracellular matrix molecules, display abnormalities of synaptic structure and function and severe deficits in perineuronal net formation.</a> J Neurosci. 33, 7742-7755 (2013).</li><li>Dzyubenko, E., Gottschling, C., Faissner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuron-Glia+Interactions+in+Neural+Plasticity:+Contributions+of+Neural+Extracellular+Matrix+and+Perineuronal+Nets.">Neuron-Glia Interactions in Neural Plasticity: Contributions of Neural Extracellular Matrix and Perineuronal Nets.</a> Neural Plast. 2016, 5214961 (2016).</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>Pyka, 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=Chondroitin+sulfate+proteoglycans+regulate+astrocyte-dependent+synaptogenesis+and+modulate+synaptic+activity+in+primary+embryonic+hippocampal+neurons.">Chondroitin sulfate proteoglycans regulate astrocyte-dependent synaptogenesis and modulate synaptic activity in primary embryonic hippocampal neurons.</a> Eur J Neurosci. 33, 2187-2202 (2011).</li><li>Eroglu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+astrocyte-secreted+matricellular+proteins+in+central+nervous+system+development+and+function.">The role of astrocyte-secreted matricellular proteins in central nervous system development and function.</a> J Cell Commun Signal. 3, 167-176 (2009).</li><li>Ethell, I. M., Ethell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinases+in+brain+development+and+remodeling:+synaptic+functions+and+targets.">Matrix metalloproteinases in brain development and remodeling: synaptic functions and targets.</a> J Neurosci Res. 85, 2813-2823 (2007).</li><li>Theocharidis, U., Long, K., ffrench-Constant, C., Faissner, A., Dityatev, A. l. e. x. a. n. d. e. r., Wehrle-Haller, B. e. r. n. h. a. r. d., Asla, P. i. t. k. &. #. 2. 2. 8. ;. n. e. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Prog Brain Res. 214, 3-28 (2014).</li><li>Dityatev, A., Rusakov, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+signals+of+plasticity+at+the+tetrapartite+synapse.">Molecular signals of plasticity at the tetrapartite synapse.</a> Curr Opin Neurobiol. 21, 353-359 (2011).</li><li>Ippolito, D. M., Eroglu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+synapses:+an+immunocytochemistry-based+assay+to+quantify+synapse.">Quantifying synapses: an immunocytochemistry-based assay to quantify synapse.</a> JoVe. , (2010).</li></ol>

The authors have nothing to disclose.

当前协议的主要目的是完全独立的神经元和星形细胞培养物，同时保持它们在共享介质。由于这个原因，所得到的培养物的纯度应在过程的开始进行验证。我们推荐使用神经元特异性微管蛋白，神经丝或蛋白质的NeuN神经元的标记，GFAP作为星形细胞标记，O4抗原少突胶质细胞前体标记，并Iba1蛋白识别小胶质细胞。 执行该协议的一个关键步骤的时候要特别注意，如由步骤说明之...

纵观几十年来，神经胶质细胞功能的一般性解释已经从一个单纯的支持对有关神经功能1积极调节作用的归属演变。因为对健康和疾病的2脑稳态其突出的影响，星形胶质细胞是科学界特别感兴趣的。在过去的几年中，研究一个多样性集中于神经胶质细胞的相互作用中体内和体外 3。然而，大多数的培养系统的不允许这两种细胞类型的单独分析和各自secretomes的。 有几种方法利用神经元和神经胶质细胞的直接共培养，实现长期持久的生存与生理有关的神经网络的发展4-6。本协议达到同样的目标，同时保持物理上分离的7两种细胞类型。相比conditioneD培养基接近8,9，我们的系统可以让研究神经细胞和星形胶质细胞之间的双向通信。分泌的信号分子的表达可以在细胞中的共享介质maturate进行监测。这个机会是特别相关的，因为星形细胞释放可溶性因子，如细胞因子，生长因子和细胞外基质分子10,11，从而调节神经元生长和功能7,12。因此，已经证明，另外在体外血小板反应蛋白视网膜神经节细胞的诱导突触13的形成。然而，其他未知因素是必要的，以使突触功能13。此外，通过星形胶质细胞释放分子具有以了解神经胶质细胞的相互作用的基础上被识别。 从小鼠和大鼠原代神经元和星形胶质细胞的培养先前已14-16所述。在这里，我们提出了一种优雅和多功能的工具，这两种细胞类型中的一种间接共培养方法结合起来。由于两个培养物物理分离但共享相同的介质，神经元，星形细胞和可溶性分子的影响，可以分别进行分析，从而为神经胶质细胞的相互作用的研究的强有力的工具。

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>B27</td><td>Gibco (Life Technologies)</td><td>17504-044</td><td></td></tr><tr><td>Cell-culture-grade water</td><td>MilliQ</td><td></td><td></td></tr><tr><td>Cytosine-&amp;#946;-D-arabinofuranoside (AraC)</td><td>Sigma-Aldrich</td><td>C1768</td><td>CAUTION: H317, H361</td></tr><tr><td>DMEM</td><td>Gibco (Life Technologies)&amp;nbsp;</td><td>41966-029</td><td></td></tr><tr><td>DNAse</td><td>Worthington</td><td>LS002007</td><td></td></tr><tr><td>Gentamycin</td><td>Sigma-Aldrich</td><td>G1397</td><td>CAUTION: H317-334</td></tr><tr><td>Glucose</td><td>Serva</td><td>22700</td><td></td></tr><tr><td>HBSS</td><td>Gibco (Life Technologies)</td><td>14170-088</td><td></td></tr><tr><td>HEPES</td><td>Gibco (Life Technologies)</td><td>15630-056</td><td></td></tr><tr><td>Horse serum</td><td>Biochrom AG</td><td>S9135</td><td></td></tr><tr><td>L-Cysteine</td><td>Sigma-Aldrich</td><td>C-2529</td><td></td></tr><tr><td>MEM</td><td>Gibco (Life Technologies)</td><td>31095-029</td><td></td></tr><tr><td>Ovalbumin</td><td>Sigma-Aldrich</td><td>A7641</td><td>CAUTION: H334</td></tr><tr><td>Papain</td><td>Worthington</td><td>3126</td><td></td></tr><tr><td>PBS</td><td>self-made</td><td></td><td></td></tr><tr><td>Poly-D-lysine</td><td>Sigma-Aldrich</td><td>P0899</td><td></td></tr><tr><td>Poly-L-ornithine</td><td>Sigma-Aldrich</td><td>P3655</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>S8636</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Gibco (Life Technologies)</td><td>25300054</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>24-well-plates</td><td>Thermoscientific/Nunc</td><td>142475</td><td></td></tr><tr><td>24-wells-plate (for the&amp;nbsp; indirect co-culture)</td><td>BD Falcon</td><td>353504</td><td></td></tr><tr><td>Binocular</td><td>Leica</td><td>MZ6</td><td></td></tr><tr><td>Cell-culture inserts</td><td>BD Falcon</td><td>353095</td><td></td></tr><tr><td>Centrifuge</td><td>Heraeus</td><td>Multifuge 3S-R</td><td></td></tr><tr><td>Counting Chamber</td><td>Marienfeld</td><td>650010</td><td></td></tr><tr><td>Forceps</td><td>FST Dumont (#5)</td><td>11254-20</td><td></td></tr><tr><td>glass cover slips (12&amp;nbsp;mm)</td><td>Carl Roth (Menzel- Gl&amp;auml;ser)</td><td>P231.1</td><td></td></tr><tr><td>Incubator</td><td>Thermo Scientific</td><td>Heracell 240i</td><td></td></tr><tr><td>Micro-tube (2 mL)</td><td>Sarstedt</td><td>72,691</td><td></td></tr><tr><td>Microscope</td><td>Leica</td><td>DMIL</td><td></td></tr><tr><td>Millex Syringe-driven filter unit</td><td>Millipore</td><td>SLGV013SL</td><td></td></tr><tr><td>Orbital shaker</td><td>New Brunswick Scientific</td><td>Innova 4000</td><td></td></tr><tr><td>Parafilm</td><td>Bemis</td><td>PM-996</td><td></td></tr><tr><td>Petri dishes (10 cm)</td><td>Sarstedt</td><td>833,902</td><td></td></tr><tr><td>pipette (1 mL)</td><td>Gilson</td><td>Pipetman 1000</td><td></td></tr><tr><td>Sterile work bench</td><td>The Baker Company</td><td>Laminar Flow SterilGARD III</td><td></td></tr><tr><td>Surgical scissors</td><td>FST Dumont</td><td>14094-11</td><td></td></tr><tr><td>Syringe</td><td>Henry Schein</td><td>9003016</td><td></td></tr><tr><td>T75 flask</td><td>Sarstedt</td><td>833,911,002</td><td></td></tr><tr><td>tube (15 mL)</td><td>Sarstedt</td><td>64,554,502</td><td></td></tr><tr><td>Water bath</td><td>GFL</td><td>Water bath type 1004</td><td></td></tr></tbody></table>

the indirect neuron-astrocyte coculture assay: an in vitro set-up for the detailed investigation of neuron-glia interactions

正确的神经细胞的发育和功能是发展中国家和成人大脑的先决条件。然而，复杂的神经元网络的高度控制形成和维护背后的机制尚未完全迄今理解。关于健康和疾病的神经元的开放式问题是多种多样的，从理解基本的发展，人类的调查相关疾病， 如阿尔茨海默氏症和精神分裂症深远。可以在体外进行的神经元的最详细的分析。然而，神经元细胞，要求和需要为他们的长期生存的额外支持星形胶质细胞。此细胞异质性是在与目标解剖神经元和星形胶质细胞的分析冲突。我们在这里提出一个细胞培养测定法，其允许纯原代神经元和星形胶质细胞，其共享相同的化学上确定的培养基的长期共培养，而在物理上分开。在此设置中，培养物存活多达四个星期，该测定是适于关于神经元的神经胶质细胞的相互作用研究的多样性。

通过间接共培养系统中的神经元培养物的分析是多方面的，可以在培养的成熟的不同阶段进行。由于这样的事实，该细胞可被保持长达4周，培养物的长期的调查是可能的。 在图1中的中间左侧面板概略说明了共培养设置。与使用该系统，可以执行这两种细胞类型的活细胞成像，由星形胶质细胞单层（左上面板）和?...

Watch this Scientific Journal Video about The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions at JoVE.com

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

该协议描述了神经元的神经胶质细胞相互作用的条块分析间接神经元的星形胶质细胞共培养。

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 ...

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Research

JoVE Journal

Neuroscience

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

14.6K 次观看.  Ruhr-University. 该协议描述了神经元的神经胶质细胞相互作用的条块分析间接神经元的星形胶质细胞共培养。 

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

Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method

In the central nervous system of all mammals, severed axons after injury are unable to regenerate to their original targets and functional recovery is very poor 1. The failure of axon regeneration is a combined result of several factors including the hostile glial cell environment, inhibitory myelin related molecules and decreased intrinsic neuron regenerative capacity 2. Astrocytes are the most predominant glial cell type in central nervous system and play important role in axon functions under physiology and pathology conditions 3. Contrast to the homologous oligodendrocytes, astrocytes are a heterogeneous cell population composed by different astrocyte subpopulations with diverse morphologies and gene expression 4. The functional significance of this heterogeneity, such as their influences on axon growth, is largely unknown.
To study the glial cell, especially the function of astrocyte heterogeneity in neuron behavior, we established a new method by co-culturing high purified dorsal root ganglia neurons with glial cells obtained from the rat cortex. By this technique, we were able to directly compare neuron adhesion and axon growth on different astrocytes subpopulations under the same condition.
In this report, we give the detailed protocol of this method for astrocytes isolation and culture, dorsal root ganglia neurons isolation and purification, and the co-culture of DRG neurons with astrocytes. This method could also be extended to other brain regions to study cellular or regional specific interaction between neurons and glial cells.

In this protocol, we described a new method to study the influence of glial cell heterogeneity on axon growth with an in vitro co-culture system. Rat cortical glial cells were cultured to confluence and cocultured with highly purified rat dorsal root ganglia neurons. Different glial cell influence on neurons adhesion and axon growth was compared directly in the same culture. This method provides a new way to directly study the glial cell heterogeneity influence on neuron adhesion and axon growth.

研究胶质细胞轴突的生长异质性的影响，使用一个新的共培养法

In the central nervous system of all mammals, severed axons after injury are unable to regenerate to their original targets and functional recovery is ...

科研

神经科学

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

二层共培养小学的大鼠皮层神经元和神经胶质

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

生物学

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2 . Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e., axon, dendrite, or soma)3. This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell.
	In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.

神经元对神经胶质细胞在微流控文化互动平台（MCP）的神经元轴突和神经胶质细胞共培养系统的影像学分析

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying the human brain. One emerging technology to address this issue is the use of three dimensional (3D) neural cell cultures, termed &#39;organoids&#39; or &#39;spheroids&#39;, for long term preservation of intercellular interactions including extracellular adhesion molecules. However, these culture systems are time consuming and not systematically generated. Here, we detail a method to rapidly and consistently produce 3D cocultures of neurons and astrocytes from human pluripotent stem cells. First, pre-differentiated astrocytes and neuronal progenitors are dissociated and counted. Next, cells are combined in sphere-forming dishes with a Rho-Kinase inhibitor and at specific ratios to produce spheres of reproducible size. After several weeks of culture as floating spheres, cocultures (&#39;asteroids&#39;) are finally sectioned for immunostaining or plated upon multielectrode arrays to measure synaptic density and strength. In general, it is expected that this protocol will yield 3D neural spheres that display mature cell-type restricted markers, form functional synapses, and exhibit spontaneous synaptic network burst activity. Together, this system permits drug screening and investigations into mechanisms of disease in a more suitable model compared to monolayer cultures.

In this protocol, we aim to describe a reproducible method for combining dissociated human pluripotent stem cell derived neurons and astrocytes together into 3D sphere cocultures, maintaining these spheres in free floating conditions, and subsequently measuring synaptic circuit activity of the spheres with immunoanalysis and multielectrode array recordings.

3D Cocultures 星形胶质细胞与人多潜能干细胞神经元的突触电路建模

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying ...

发育生物学

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.
To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.
To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

研究神经-胶质相互作用在缺血中的作用的细胞培养模型

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...

 用于 CNS 相互作用研究的原代小胶质细胞和神经元共培养方案

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS homeostasis. They are a major population of immune cells associated with CNS disease activity, adopting&#160;reactive phenotypes that potentially contribute to neuronal injury during chronic neurodegenerative diseases such as multiple sclerosis (MS). The distinct mechanisms by which microglia regulate neuronal function and survival during health and disease remain limited due to challenges in resolving the complex in vivo interactions between microglia, neurons, and other CNS environmental factors. Thus, the in vitro approach of co-culturing microglia and neurons remains a valuable tool for studying microglia-neuronal interactions. Here, we present a protocol to generate and co-culture primary microglia and neurons from mice. Specifically, microglia were isolated after 9-10 days in vitro from a mixed glia culture established from brain homogenates derived from neonatal mice between post-natal days 0-2. Neuronal cells were isolated from brain cortices of mouse embryos between embryonic days 16-18. After 4-5 days in vitro, neuronal cells were seeded in 96-well plates, followed by the addition of microglia to form the co-culture. Careful timing is critical for this protocol as both cell types need to reach experimental maturity to establish the co-culture. Overall, this co-culture can be useful for studying microglia-neuron interactions and can provide multiple readouts, including immunofluorescence microscopy, live imaging, as well as RNA and protein assays.

作者聚焦:用于研究疾病条件下小胶质细胞-神经元相互作用的体外共培养模型

This protocol describes a microglia-neuronal co-culture established from primary neuronal cells isolated from mouse embryos at embryonic days 15-16 and primary microglia generated from the brains of neonatal mice at post-natal days 1-2.

生成和共培养小鼠原代小胶质细胞和皮质神经元

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS ...

An Indirect Neuron-Astrocyte Co-Culture Technique for Studying Neuron-Glia Interactions

Source: Gottschling, C. et al., <a href="https://app.jove.com/v/54757">The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions.</a>&#160;J. Vis. Exp.&#160;(2016)
This video illustrates a method for establishing an indirect neuron-astrocyte co-culture using a compartmentalized approach. In this setup, neurons and astrocytes are spatially separated but share a conditioned medium. Neurotrophic factors released by astrocytes promote neuron differentiation and the formation of synaptic connections, suggesting neuron-glia interactions.

一种用于研究神经元-胶质细胞相互作用的间接神经元-星形胶质细胞共培养技术

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

间接神经元的星形胶质细胞共培养试验:一个

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此视频中的章节

研究胶质细胞轴突的生长异质性的影响，使用一个新的共培养法

二层共培养小学的大鼠皮层神经元和神经胶质

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

神经元对神经胶质细胞在微流控文化互动平台（MCP）的神经元轴突和神经胶质细胞共培养系统的影像学分析

3D Cocultures 星形胶质细胞与人多潜能干细胞神经元的突触电路建模

研究神经-胶质相互作用在缺血中的作用的细胞培养模型

生成和共培养小鼠原代小胶质细胞和皮质神经元

生成和共培养小鼠原代小胶质细胞和皮质神经元

生成和共培养小鼠原代小胶质细胞和皮质神经元

一种用于研究神经元-胶质细胞相互作用的间接神经元-星形胶质细胞共培养技术