所有的程序获得批准委员会威斯康星大学动物护理，并按照美国国立卫生研究院的指引。我们感谢她Nucleofector设备的大量使用凯瑟琳Kalil博士。我们也感谢登特实验室成员协议的意见。这项工作是支持由NIH R01 - NS064014，达纳基金会和白厅基金会的赠款，以EWD 克里Viesselmann，贾森Ballweg和德里克Lumbard同等贡献本文。

1。盖玻片和分庭的制备<ol><li>干净的盖玻片和商会的制备是健康的文化所必需的。快捷键不应该采取任何这些步骤。 </li><li>洗净的盖玻片（12mm或22毫米轮，德国玻璃 - 卡罗来纳州助理品牌）集中在一个专用的玻璃瓶或烧杯中的硝酸（HNO3）过夜。 </li><li>从硝酸中删除的盖玻片，在去离子水冲洗广泛（5 7X）。 </li><li>在层流罩或生物安全柜的盖玻片和干分开。干燥时，消毒30​​分钟的紫外线光。消毒的无菌培养皿储存菜盖玻片。如果12毫米盖玻片电镀神经元直接放置在无菌的35mm盘的盖玻片，进行第2节。 </li><li>影像室，钻一个孔（删除所有毛刺）的35mm培养皿的底部15毫米和附加清洁盖玻片，用3:1的石蜡和凡士林混合构造。 </li><li>熔体在锥管内沸水浴的石蜡/凡士林混合物。使用一个小的油漆刷和大衣盘周围的15毫米洞下方。请一定要保持石蜡/凡士林混合搅拌，因为它会分开。这通常会导致与凡士林的浓度较高，这将成为粘稠的菜肴放置在孵化器，在盖玻片支队粘在一起商会。其余石蜡/凡士林可以在室温下保存。 </li><li>将菜倒放在一个平面托盘，并将其放置在孔盖玻片。 80 ° C烘箱中加热，直到石蜡混合物融化（大约10分钟）。取出一个平坦的表面上的菜肴，让石蜡混合物集。 </li><li>打开菜，消毒的紫外灯盖和底部商会内部。 </li><li>大衣盖玻片或地区的商会，1.0mg/mL聚- D -赖氨酸在硼酸盐缓冲液（0.1M硼酸钠，pH值8.5）（30kDa）一小时的玻璃。丰富的组织文化品位去离子水冲洗3-5次。确保删除所有的硼酸盐缓冲的痕迹。干燥和使用即时或存储商会/盖玻片，以供日后使用。我们通常使用的准备工作后一个月内清洗盖玻片。 </li></ol> 2。神经解剖和培养基的制备<ol><li>准备组织文化品位水通过添加适当数量的10倍HBSS和100倍的HEPES的夹层介质（DM）。保存在4 ° C。在冰上保持在清扫。 </li><li>解剖前一天准备电镀液（下午）和无血清培养基（SFM）。下午由Neurobasal中等，B27的补充，2毫米谷氨酰胺​​，0.3％的葡萄糖，氯化钠37.5毫米和5％胎牛血清（FBS），。 SFM Neurobasal中等，B27的补充，谷氨酰胺2毫米，0.3％葡萄糖和37.5毫米氯化钠。 </li><li>只在组织文化的清扫和存储足够的孵化器与盖隔夜稍微虚掩着，使温度和CO 2的介质达到平衡的内容。我们增加了额外的葡萄糖和渗透压增加，氯化钠约310mOsm。我们找到的文化做一个更生理的渗透压（Neurobasal渗透压一般为205 - 245mOsm）更好。 </li></ol> 3。长期培养的皮层神经胶质直属层的制备<ol><li>如果要准备长期的文化，执行这部分的协议的两至三个星期，然后再继续与皮质或海马解剖。 </li><li>准备胶质培养基与MEM（GM）0.3％的葡萄糖，青霉素/链霉素和10％马血清。 </li><li>安乐死P1 - P3的小鼠的幼崽在冰上冷却5分钟。从冰和喷用70％乙醇中删除每个小狗。用剪刀快速斩首。整个大脑取出一盘冷DM（步骤2.1）。 </li><li>移除大脑两半球和脑膜。海关新皮层，并转移到一个新的菜，不包含任何媒体。准备共4大脑皮质。 </li><li>尽可能细切碎，用干净，无菌刀片皮层，并取出用塑料吸管50 mL锥形管，含有12毫升的冷DM切碎组织。添加胰蛋白酶和DNase 0.25％（1.5毫升）和0.1％（1.5毫升），终浓度。在37℃水浴孵育10分钟间歇纷飞。 </li><li>取出试管中皮层组织，用70％乙醇引入到组织培养罩前要彻底清洁。吸取皮质组织与一个10毫升吸管最块约10-15次，或直至消失。 </li><li>返回管至37 ° C，在水浴10分钟间歇纷飞。 </li><li>彻底清洁，用70％乙醇管，并把它带回去的组织文化罩。吸取的皮层组织和5米大号吸管约10-15次，或直至块消失。 </li><li>添加15毫升的温暖通用和200xg（1000RPM）离心10分钟。 </li><li>弃上清，重悬在20毫升的新鲜GM颗粒细胞，并与一个血球计数。板5 - 7.5x10 6细胞15毫升每75厘米2烧瓶总经理。 </li><li>一天后，每2-3在随后的日子里文化，打跑松散的细胞，对你的手敲烧瓶。删除任何脱落细胞中，并与15毫升的新鲜GM替换。 </li><li>神经胶质细胞可以收获后1-2周在烧瓶增长，当他们约70-100％融合。为了准备与神经胶质涂层的个人盖玻片，放置6硝酸清洗和消毒，在10厘米菜25毫米圆形盖玻片，并将其放置在一个三角形图案用小油漆刷到每个盖玻片3点3:1混合石蜡/凡士林。 30分钟的紫外线光对开放的菜肴。大衣与0.1 mg / mL的聚- D -赖氨酸在硼酸盐缓冲液（30kDa）一小时，然后洗净广泛用无菌组织的文化品位去离子水（3 - 5倍），并让干燥的盖玻片。 </li><li>从孵化器中取出含有胶质烧瓶，弃培养基预热胰蛋白酶/ EDTA溶液5毫升冲洗。从烧瓶中取出的胰蛋白酶/ EDTA溶液和3毫升新鲜预热胰蛋白酶/ EDTA吸管入烧瓶。在37℃前加入5毫升的GM停止胰蛋白酶为1分钟瓶。 </li><li>从烧瓶中取出的神经胶质细胞，反复吹打10-15次，然后媒体转移到15 mL锥形管。 200xg（1000RPM）离心8分钟。去除上清液，并添加12.5毫升每10厘米菜含有盖玻片的GM通用汽车的10毫升，计数细胞， 板 5X10 5细胞。 </li><li>每2-3天在新鲜预热GM交换介质。神经清扫的前一天，通用汽车公司和与SFM（2.2节）更换。皮质或海马文化水浸时，使用在步骤4.12胶质空调的可持续森林管理。 </li></ol> 4。皮层和/或海马解剖和电穿孔<ol><li>删除适当数额的nucleofection解决方案（龙沙），结合起来，温暖的室温开始剥离之前。由于Nucleofection解决方案的有限生命周期相结合时，我们只有结合每个准备（100μL每转）所需的金额。 </li><li>安乐死在E15.5怀孕的老鼠与CO 2（插件一天E0.5）和一个10cm的培养皿中取出子宫。取出胎儿和斩首，到寒冷的DM（2.1节）。 </li><li>取出到一个单独菜冷DM的整个大脑和弯曲的钨针，同时删除neocortices。删除新菜冷DM microforceps和地方皮层脑膜。虹膜或老爷车用小剪刀，删除在1.5毫升的Eppendorf管充满了1.0毫升的冷DM皮质或海马和地点。此管置于冰上。 </li><li>所有皮质或海马解剖后，加110μL2.5％胰蛋白酶的Eppendorf管中，包含在37℃培养箱20分钟的组织和地方管。 </li><li>轻轻颠倒Eppendorf管中上清，洗皮质或海马取出1.0毫升下午（2.2节）。重复洗两次澡，留在管1毫升的PM。 </li><li>磨碎的块15倍，与P1000的吸管，除去上清液/细胞到一个新的15 mL锥形管中含有4毫升的PM，留在Eppendorf管中仍然存在的任何块。 </li><li> 7分钟关闭制动20xg（350rpm）旋转15毫升管。弃上清，每转增加100μL预混，室温nucleofection解决方案（龙沙）。磨碎的P1000的吸管轻轻向上和向下运动的5倍。 </li><li>每一个新的Eppendorf管中取出100μLnucleofection解决方案/细胞混合，并添加适量的DNA。对于长期的文化，我们一般采用1 -2μg每转染的DNA。然而，这一个小的文化中的神经元的比例&lt;10％的标签。我们一般使用每转5 -10μg的DNA，如果想短期文化的转染效率更高。我们使用的40μg的DNA的总时与两个不同的质粒转染。质粒存储在1μg/μLTE缓冲液。 </li><li>加入细胞悬液/ DNA的比色皿（龙沙）和electroporate Nucleofector（龙沙）的细胞，使用程序O - 005（小鼠中枢神经系统的神经元）。 </li><li>工作快速，加500μL预热和平衡下午试管和删除的解决方案/一个新的1.5 mL Eppendorf管的细胞。补充足够的下午带来的每转的体积为1.0 mL。一个血细胞计数板计数细胞在3 - 5X10 3细胞/长期文化对青少年的文化，或5 - 10 × 10 3细胞/ 厘米2厘米2 。/ LI&gt; <li>对于短期文化，洪水2.0毫升的温暖，CO 2平衡可持续森林管理的电镀一小时后的35毫米培养皿。如果使用盖玻片，我们删除的PM的一半，替换与SFM，然后重复两次。泛滥的成像商会或洗涤盖玻片结果在一个非常低的血清含量（&lt;0.5％）。短期文化不需要与胶质细胞滋养层培养，不需要再喂。 </li><li>对于长期的文化，我们删除一个神经胶质细胞盖盖玻片，包含三个点的石蜡/凡士林和15毫米孔在35mm盘，初步电镀后一小时以上反转。从胶质菜空调的SFM毫升，然后添加到成像室。饲料长期的文化，我们的可持续森林管理，每2-3天中删除的三分之一，代之以新鲜， 预热和CO 2平衡可持续森林管理。 </li></ol> 5。代表性的成果: <img src="/files/ftp_upload/2373/2373fig1.jpg" alt="图1" /> 图1。生活海马神经元在连续的发展阶段。配对生活代表的海马神经元的图像显示为微分干涉对比度的图像和相应的荧光显微镜。这些细胞已pCAX向量与EGFP -微管蛋白和DsRed2转。在随后的日子里在体外 （DIV）:第1阶段（1DIV），第2阶段（1DIV），第3阶段（2DIV），第四阶段（11DIV）和第五阶段（32DIV）成像神经元。比例尺是20微米。

<ol>
	<li>Goslin, K., Asmussen, H., Banker, G., Goslin, K., 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=Chapter+13.">Chapter 13.</a> Culturing Nerve Cells. , 339-370 (1998).</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>Dent, E. W., Callaway, J. L., Szebenyi, G., Baas, P. W., Kalil, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reorganization+and+movement+of+microtubules+in+axonal+growth+cones+and+developing+interstitial+branches.">Reorganization and movement of microtubules in axonal growth cones and developing interstitial branches.</a> J Neurosci. 19, 8894-8908 (1999).</li><li>Dent, E. W., Kalil, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+imaging+of+neuronal+cytoskeleton.">Dynamic imaging of neuronal cytoskeleton.</a> Methods Enzymol. 361, 390-407 (2003).</li><li>Dent, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filopodia+are+required+for+cortical+neurite+initiation.">Filopodia are required for cortical neurite initiation.</a> Nat Cell Biol. 9, 1347-1359 (2007).</li><li>Hu, X., Viesselmann, C., Nam, S., Merriam, E., Dent, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-dependent+dynamic+microtubule+invasion+of+dendritic+spines.">Activity-dependent dynamic microtubule invasion of dendritic spines.</a> J Neurosci. 28, 13094-13105 (2008).</li><li>Lebrand, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+role+of+Ena/VASP+proteins+for+filopodia+formation+in+neurons+and+in+function+downstream+of+netrin-1.">Critical role of Ena/VASP proteins for filopodia formation in neurons and in function downstream of netrin-1.</a> Neuron. 42, 37-49 (2004).</li><li>Meberg, P. J., Miller, M. W., Hollenbeck, P. J., Bamburg, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+7.">Chapter 7.</a> Neurons: Methods and Applications for the Cell Biologist. , 112-129 (2003).</li><li>Osumi, N., Inoue, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+into+cultured+mammalian+embryos+by+electroporation.">Gene transfer into cultured mammalian embryos by electroporation.</a> Methods. 24, 35-42 (2001).</li><li>Zeitelhofer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+transfection+of+mammalian+neurons+via+nucleofection.">High-efficiency transfection of mammalian neurons via nucleofection.</a> Nat Protoc. 2, 1692-1704 (2007).</li></ol>

作为一个银行家协议，它使用大鼠神经元1,2的修改，这为培养的胚胎海马和皮质鼠标神经元的协议。我们已经用3,4,5,6,7，以及为培养小鼠和仓鼠神经元这一协议。该协议同样适用海马和新皮层神经元和类似Meberg和米勒8出版的协议。一般来说，我们使用长期培养的海马神经元，因为他们是很好的特点和更成熟的模型系统。此外，它们可能含有更均匀的人口比大脑皮层的神经元。然而，新皮层神经元的培养，使用此协议，也同样生存和分化（未发表数据）。海马和新皮层神经元的短期文化，我们经常使用。大脑皮层夹层结果比海马夹层（2.5 × 10 5％海马对神经元），这使得它的材料更好的选择免疫印迹例如，更多的神经元（1.5x10 6个神经元皮质对）。 任何小学文化，它是必不可少的，以尽量减少所需的时间从死亡的动物细胞电镀。它通常会采取1​​0-20解剖成为始终在快速剥离和电镀。此外，龙沙Nucleofector工作时，它是关键在电过程迅速，作为神经元的活力迅速下降，如果他们都留在nucleofection缓冲区。 我们的成像与总内部反射荧光显微镜（TIRFM）进行。这种类型的显微镜只能够成像几百纳米超出盖玻片。因此，神经元的地区，我们经常形象，轴突的生长锥和树突棘，需要坚持直接盖玻片。因此，我们使用低密度的文化，需要长期培养的神经胶质喂养。我们已经使用了密度较高的文化（&gt; 2 × 10 4细胞/ 厘米 2），没有胶质细胞的饲养层，长期培养，发现他们很少喂养很好的生存。然而，这些神经元的树突棘常常过于远离基板TIRFM形象，虽然他们可以很容易地与宽视场显微镜或共聚焦显微镜检测。 在大多数我们的研究，我们转染电镀前的神经元，并有高达文化三个月成像荧光标记的蛋白质。这种荧光标记的蛋白质长期的表现，使用低浓度的DNA（1 -2μg），我们不生产过度表达在神经元的文物给了我们信心。但是，这个过程也可以被用于研究蛋白质的过度表达，如果使用高量的DNA（10 -20μg）。我们使用的质粒，转染神经元通常含有EGFP或mCherry融合蛋白，虽然我们也标签DsRed2或EGFP单独的神经元的胞浆。这电技术的工作原理以及与向量的数量。我们宁愿含有β-肌动蛋白启动子与CMV增强子和β-珠蛋白的质粒，聚尾巴（pCAGGs或pCAX质粒）9，由于相对较高的水平表达，而他们是由神经元的耐受性良好在短期和长期的文化。一般来说，蛋白质在约4小时电镀内开始表达，达到成像足够的水平在10 - 24小时10。我们已成功地用于巨细胞病毒启动子驱动的质粒在短期文化，但发现它们可能会导致高层次的过度表达，杀死神经细胞长期培养。不过，我们已经发现，低密度培养的神经胶质空调帮助与​​巨细胞病毒启动子驱动的质粒转染神经元的生存，而密度较高的（非胶质美联储）文化。

*大部分储存于-80 ° C，可在-20 ° C储存以及试剂。储存在-80 ° C延长其保质期和稍微比较一致的文化的结果。

nucleofection and primary culture of embryonic mouse hippocampal and cortical neurons

海马和皮层神经元已被广泛地用于研究中枢神经系统（CNS）神经细胞极化，轴突/枝晶生长，突触的形成和功能。培养这些神经元的一个优点是，他们很容易分化，形成独特的轴突和树突，在非常低的密度上的两个立体基板。此属性，使他们非常确定神经元发育的许多方面作出了有益的。此外，通过提供这些神经元的神经胶质空调，他们将继续发展，形成功能性突触连接，尚存在几个月文化。在这个协议中，我们勾勒出一种技术，解剖，文化和转染小鼠胚胎海马和皮层神经元。转染是通过电穿孔进入神经元的DNA，然后通过nucleofection电镀。此协议已经表达荧光标记的融合蛋白的早期发展（〜4 8小时后镀），在两极分化，轴突的生长和分支研究的动态与蛋白质的功能优势。我们还发现，这在电镀前的单转保持在适当的水平，整个一生的神经元（&gt; 2个月的文化）成像荧光标记的融合蛋白的表达。因此，这种方法是研究神经元功能很少或根本没有中断蛋白定位和整个中枢神经系统发育的功能非常有用。

Watch this Scientific Journal Video about Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons at JoVE.com

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

该协议概述解剖所需的步骤，通过电穿孔和文化小鼠海马和皮层神经元的转染。短期可用于文化研究轴突的生长和指导，而长期的文化可以为研究突触和树突棘分析。

Nucleofection和胚胎小鼠海马和皮层神经元的原代培养

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

nucleofection-primary-culture-embryonic-mouse-hippocampal-cortical

Nanyang Technological University

Research

JoVE Journal

Neuroscience

33.3K 次观看.  University of Wisconsin-Madison. 该协议概述解剖所需的步骤，通过电穿孔和文化小鼠海马和皮层神经元的转染。短期可用于文化研究轴突的生长和指导，而长期的文化可以为研究突触和树突棘分析。

视频: Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

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

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

在子宫内的电穿孔其次原发性神经文化为研究基因的功能在皮层神经元的一个子集

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons1. Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract2,3. More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)4. Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75NTR. The p75NTR can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75NTR is exclusively expressed by the spinal motoneurons5. This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells6. Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75NTR has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75NTR as well7. The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75NTR antibody. The lectin is an extremely inexpensive alternative to the p75NTR antibody and the purification grades using lectin are comparable to that of the p75NTR antibody. Motoneurons from the embryonic spinal cord can be isolated by this method, survive and grow out neurites.

An alternative way of isolating mouse embryonic motoneurons from the spinal cord is described. The method takes into account the fact that lectin can bind to the low affinity nerve growth factor receptor p75NTR. This lectin-based preplating allows a purification similar to that with a specific antibody against the p75NTR.

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. ...

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 ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

从产前小鼠海马神经元的分离和培养

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.

Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.

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 ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

原代海马神经元的磷酸钙转染

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

小鼠多巴胺能神经元的原代培养

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

原发性脑多巴胺能神经元的分离，培养和长期维持胚胎脑鼠类

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Low-Density Primary Hippocampal Neuron Culture

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This &#39;sandwich culture&#39; allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

低密度原海马神经元文化

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...