Name: low-density primary hippocampal neuron culture
Uploaded: 2017-04-18T12:30:00.000+00:00
Duration: 17 min 46 s
Description: Watch this Scientific Journal Video about Low-Density Primary Hippocampal Neuron Culture at JoVE.com

这项工作是由CIHR MOP-142209到TJS支持。

所有实验和使用实验动物方案由曼尼托巴大学动物伦理委员会批准并且是符合加拿大动物保护协会的指导方针。 1.仪器，缓冲液和溶液的制备<ol><li>通过高压灭菌所有解剖设备，玻璃巴斯德移液管，移液管尖端，过滤装置，和去离子水进行消毒。 </li><li>制备20％（w / v的1.1 M）在去离子水和过滤灭菌库存葡萄糖溶液。储存于4℃。 </li><li>制备在去离子水和过滤灭菌100mM的丙酮酸钠溶液。储存于4℃。 </li><li>制备含钙和镁的Hank氏平衡盐溶液（CMF-HBSS）通过使10％的HBSS（10倍），10mM的HEPES，和100U / mL的青霉素 - 链霉素的在去离子水中的溶液。存储在4℃下不超过1至2个月。 </li><li>制备10毫克/毫升脱氧核糖核酸酶I（DNA酶）在CMF-HBSS，然后过滤灭菌，并在-20℃下储存。 </li><li>制备通过使30mM葡萄糖的溶液（来自无菌溶液）胶质介质，95 U / ml的青霉素 - 链霉素，和10％-15％牛生长在最低必需培养基（MEM）血清（BGS）。 注意:作为在所述协议指示的，如果需要减缓细胞的增殖，可以使用5％的BGS的溶液。马血清可被用来代替BGS，但要注意，每一批次都必须在使用前，由于批次间的变化进行测试。存储在4℃下制备的溶液为不超过1至2个月。尽管许多实验室使用FBS，我们始终认为，在BGS神经胶质细胞生长是一样强大的FBS。 BGS是由补充有化学上确定的组分胎牛血清的。此外，BGS实质上比FBS更经济。 </li><li>通过使溶液制备神经元生长和维持培养基（的Neurobasal-B27，NBG）1X B27添加剂和0.5mM L-谷氨酰胺在500ml的Neurobasal的Medi嗯。存储在4℃下不超过1至2个月。 L-谷氨酰胺可以含GlutaMAX，已经提出的是在生长培养基中更稳定的被取代。 </li><li>制备神经元通过使30mM葡萄糖的溶液（来自无菌溶液）平板培养基，1mM丙酮酸钠（来自无菌溶液），和10％BGS在MEM中。存储在4℃下不超过1至2个月。 </li><li>制备1mM的阿糖胞苷（阿糖胞苷）的溶液中并过滤灭菌。分装成每份1ml，并储存在-20℃。 </li><li>制备的100mM的APV在100 MN过滤灭菌的NaOH溶液。存储在-20℃。 </li><li>溶解1毫克/毫升聚L-赖氨酸在硼酸盐缓冲液，pH 8.5（50mM的硼酸和12mM的硼砂）和过滤灭菌。等分试样并储存在-20℃。代替聚-L-赖氨酸的，可以使用聚L-鸟氨酸，其可能是有毒的细胞较少。 </li></ol> 2.神经胶质文化准备<ol><li>准备皮质星形胶质细胞培养<ol><li&gt;获取出生后24个小时内幼鼠。放置在冰幼崽到麻醉他们。 </li><li>喷雾用70％乙醇的幼崽和他们斩首。 </li><li>放置在磁头上含有15毫升CMF-HBSS冰100mm培养皿。 </li><li>通过切割头皮，打开头骨，切断脑干，然后将脑转移至60mm培养皿上含有3毫升CMF-HBSS冰解剖出脑的每个。 </li><li>从海马中分离大脑半球，然后剥去它们周围的脑膜。应格外注意事项，以确保从脑膜成纤维细胞污染最小。在文化脑膜成纤维细胞可以与神经胶质细胞生长竞争，也不利于对神经元的健康。收集在含有5毫升CMF-HBSS冰60毫米培养皿中的所有大脑半球。 </li><li>卸下CMF-HBSS，然后使用微解剖弹簧剪刀切碎收集的皮层组织尽可能细。 </li><li>足够的CMF-HBSS加入斩PED组织。使用玻璃巴斯德吸管，该组织片转移至15mL管中。通过添加CMF-HBSS，使总体积至12毫升。 </li><li>添加1.5mL的各2.5％胰蛋白酶和10毫克/毫升DNA酶溶液。孵育在水浴中12分钟，在37℃将得到的混合物。 </li><li>磨碎所述组织使用玻璃巴斯德吸管10-15倍，并进一步孵育它在37℃水浴中3分钟。 </li><li>过滤通过镜头纸或滤网到含有5毫升的BGS的50mL管中的上清液。这样做是为了去除未解离的组织块。另外，使用市售的细胞滤网。 </li><li>添加13.5毫升CMF-HBSS和1.5mL的2.5％胰蛋白酶的含有剩余的组织片的管中并在37℃的水浴中孵育进一步它另外的10分钟。 </li><li>磨碎剩余组织一次，然后再次过滤悬浮液到含有初始滤液中的50mL试管。接下来，冲洗乐纳秒纸用3mL BGS的。最终的体积应为大约38毫升。 </li><li>离心机含有解离的神经胶质细胞在130×g下6分钟的悬浮液中。仔细丢弃使用玻璃巴斯德吸管将上清液。确保在底部沉淀的细胞不受到干扰。的粒料的底部会出现含血液成分微带红色。 </li><li>转移在1mL胶质介质的神经胶质细胞到新的管中，注意不要扰乱粒料的微红部分。 </li><li>加起来2毫升每小狗胶质介质的重悬结合解离的神经胶质细胞。例如，如果10只幼仔被使用，然后再悬浮总体积将是20毫升。 </li><li>制备每一个小狗75cm 2的细胞培养瓶。 18毫升预热的胶质介质添加到每个烧瓶中。 </li><li>转印2mL的细胞悬浮液到每个烧瓶中并在37℃，5％CO 2培养箱中孵育。 </li><li>一天后，更换用20mL胶质米edium。在这个阶段，文化是胶质和其他不需要的细胞类型的混合。 </li><li>接种细胞四天后，大力摇晃瓶打跑非星形神经胶质细胞。要做到这一点，用CMF-HBSS冲洗烧瓶一次，然后添加10毫升新鲜CMF-HBSS到每个烧瓶中。摇瓶中大力5-10次，然后吸掉CMF-HBSS。用CMF-HBSS冲洗两次，然后添加20毫升胶质平台。 注:此步骤是为了确保消除非靶细胞类型的关键，不应该产生期望的星形胶质细胞的损失。 </li><li>与两次新鲜胶质介质每周更换直至细胞是融合（ 图1）。 </li></ol></li><li>冻结胶质<ol><li>一旦培养物汇合，吸掉培养基并用10毫升CMF-HBSS洗涤细胞单层。 </li><li>加入10 mL 0.25％胰蛋白酶/ EDTA的每个烧瓶中并在37℃下孵育3分钟。 </li><li>点击瓶轻轻打跑细胞。加入1毫升BGS吨o每个量瓶中，将细胞悬浮液到15 mL管。 </li><li>离心管，在130×g下6分钟。弃去所得的上清液。 </li><li>重悬在2mL胶质介质细胞沉淀。 </li><li>制备胶质冷冻介质（1-份二甲亚砜和4-份BGS）。添加这种溶液到每个每烧瓶4支冻存管的0.5毫升。 </li><li>转移0.5mL的细胞悬浮液到每个小瓶中，然后通过将小瓶在绝缘容器中，在-80℃冷冻器中过夜冷冻细胞慢。一天后，将小瓶转移到液氮冷冻进行长期储存。 </li></ol></li><li>电镀恢复神经胶质<ol><li>板块胶质细胞神经元文化的前一天1-2周。 </li><li>制备50mL试管用10ml温热胶质平台。 </li><li>解冻神经胶质的1个冷冻瓶在37℃水浴中2-3分钟。 </li><li>转移小瓶的内容物到含有10毫升胶质介质中的50mL试管。 </li><li>离心机E中的管在130×g离心5分钟，吸出上清液。 </li><li>重悬在20mL胶质介质沉淀。 </li><li> 3毫升新鲜胶质介质添加到每个培养皿（60mm）上，然后转移细胞悬液1mL至每个培养皿中。孵育培养物在37℃，5％CO 2培养箱中。 </li><li>订阅细胞每周两次。如果细胞密度达到50％汇合以及前神经元培养一天，切换到含有5％BGS减慢神经胶质生长速率胶质平台。对于神经元培养物中的神经胶质单层的期望的汇合率为50％ - 70％。 </li><li>替换NBG介质中的神经元培养物前1-2天的神经胶质介质（6毫升/皿）。 </li></ol></li></ol> 3.准备盖玻片<ol><li>清洁和石蜡珠的制备<ol><li>淹没盖玻片在浓硝酸（70％重量/重量;注意:高腐蚀性），并超声处理它们在室温下30分钟。其他实验室有found它有利于在孵育70％硝酸盖玻片在陶瓷机架在室温下12-24小时来代替。 </li><li>仔细丢弃根据机构指引一个指定通风橱的硝酸。冲洗盖玻片，用去离子水3-5倍。 </li><li>浸没在去离子水中的盖玻片，并再次超声处理它们30分钟（如果跳过以下的另一种方法）。 </li><li>取出去离子水和冲洗盖玻片另一三次。 </li><li>淹没在50mL去离子水将盖玻片和通过高压灭菌消毒他们。可替代地，消毒在225℃下将盖玻片在烘箱中6小时。如果使用这种替代方法，请跳到步骤3.1.9。 </li><li>一旦高压灭菌，将盖玻片转移至无菌的层流罩用于以下步骤。 </li><li>除去去离子水，并用20-30毫升100％乙醇冲洗。 </li><li>添加20-30 mL的100％乙醇并转移盖玻片和乙醇至100毫米培养皿中。 </li><li>使用钝镊子盖玻片转移至60mm培养皿中，每个培养皿4-5盖玻片，然后将它们允许风干通过倚靠盖玻片抵靠盘的边缘。一旦乙醇被蒸发，放置盖玻片的表面上平坦的。 </li><li>转移10克石蜡至合适的耐热玻璃瓶中。熔化的石蜡，并在约150-200℃下保持它的热板上至少1小时。到的石蜡应加热的理想温度可能需要一些调整。非理想的温度会导致难以生产的石蜡珠正确的尺寸。融化后的等待期可确保整个液体温度一致。 </li><li>浸巴斯德吸管插入石蜡，然后迅速它触摸到靠近每个盖玻片边缘三个点（每个点为约120°分开）。液滴将固化成珠大约0.3-0.5毫米高和1.0-2.0毫米直径ETER并且将起作用以提供盖玻片上的神经元和下面的胶质单层（ 图2）之间的空间。 </li><li>消毒在UV光下的菜30分钟，然后用铝箔和存储在室温下覆盖它们。准备盖玻片可以存储和使用长达一个月。石蜡珠应该被允许坚持以盖玻片至少一周后使用。这将防止石蜡从在协议的剩余步骤分离。 </li></ol></li><li>涂层盖玻片用聚L-赖氨酸<ol><li>得到含有与石蜡珠预先制备的盖玻片培养皿中。 </li><li>在硼酸盐缓冲液中添加多聚-L-赖氨酸的200μL到每个盖玻片的表面，并让溶液坐在盖玻片过夜，覆盖，在室温下。涂层相同的表面在其上石蜡珠位于一个。请注意，如果适当清洗，聚L-赖氨酸应该很容易蔓延以覆盖盖玻片的表面上。 </li><li>一天后，通过在无菌去离子水中浸泡他们为每次2小时洗涤盖玻片两次。最后一次洗涤后，用4mL每皿神经元电镀介质的更换水。请注意，重要的是不将盖玻片的表面被允许涂有聚-L-赖氨酸具有后干燥。 </li><li>放置含盖玻片-培养皿在37℃，5％CO 2培养箱中。将涂覆的盖玻片应温育的神经元培养物之前至少24个小时。这样做是为了黄金的神经细胞培养的培养基。 </li></ol></li></ol> 4.解剖海马和电镀神经元<ol><li>获得妊娠大鼠（18-19天胚胎，如从阴道塞发现之日测量的）和异氟醚麻醉的断头或其他批准的方法安乐死。 </li><li>喷用70％乙醇大鼠的下侧，然后从耻骨区域救援人员到场做一个切口啊到腹腔的端部。为了避免污染，照顾只有通过这一步皮肤切开。 </li><li>用70％乙醇再次冲洗解剖仪器，然后通过腹壁切开以暴露子宫。拆下两个子宫角，并放置在无菌的100毫米培养皿中。 </li><li>执行在层流通风橱中的剩余步骤。除去围绕胎儿子宫膜。斩首他们并放置头在含有20毫升CMF-HBSS 100毫米培养皿中。 </li><li>解剖出脑，并立即将它们放置在含有在冰上2毫升CMF-HBSS 60毫米培养皿中。收集每盘只有2-3大脑，使下一步足够的空间。 </li><li>在解剖显微镜下从大脑半球的中线剥去脑膜，然后解剖出海马，并将它们置于含4.5毫升CMF-HBSS 60毫米培养皿中。需要注意的是，海马可以区分作为一个月牙形STRUCTURE在皮质半球的内侧表面上，并且是易于除去，因为它是由一个心室横向界定。 </li><li>转移收集海马组织和CMF-HBSS至15mL管中。添加0.5mL的2.5％胰蛋白酶，然后在37℃下孵育10分钟。木瓜蛋白酶，可以用来代替胰蛋白酶，因为它是温和的，并可能会增加产量。 </li><li>轻轻地取出由巴斯德吸管胰蛋白酶溶液，使在该管的底部不受干扰海马组织。 </li><li>轻轻用5mL CMF-HBSS洗涤两次组织，同时在37℃下孵育其5分钟。第二次洗涤后，通过添加CMF-HBSS足够体积的组织，使总体积至少为5毫升。如果有来自10多个幼仔海马，多达8个可以被添加毫升CMF-HBSS中。 </li><li>准备组织解离火抛光移液器的两个变种。一个变体的前端是移液管，但具有火抛光平滑边缘正常直径的。其他VARI蚂蚁是一个与尖端直径减半。简言之暴露的玻璃巴斯德吸管对角于火焰源的尖端，然后检查移液器的尖端。 <ol><li>重复此步骤，直至尖端边缘已平滑化或直径已经减小。它练习找到理想的直径，以确保组织分离，获得单个细胞而不损伤这些细胞是很重要的。 </li></ol></li><li>首先由约10道次磨碎海马组织通过一个边缘光滑的正常玻璃巴斯德吸管，并通过与所述直径减小的吸液管，然后进一步的5至10次。我们的目标是获得无细胞或极少组织团块的均匀的溶液。 </li><li>确定使用血细胞计数器或自动细胞计数器中的细胞密度。典型地，从两个半球海马结合时，获得0.8至1.0百万细胞每小狗。 </li><li>传输足够的体积的细胞悬浮液，以菜含有聚-L-赖氨酸包被的盖玻片在4mL平板培养基以获得每60毫米盘300,000个细胞的接种密度，。细胞的密度可以根据预期的实验而变化从100,000至500,000。 </li><li> 3-4小时后，用连接到在含神经胶质细胞在NBG介质菜肴神经元传递的盖玻片上。应该是这样的，使得在其上的神经元铺板侧朝下朝向胶质细胞层。添加总共每胶质培养皿4-5盖玻片。 </li><li>电镀后两或三天，阿糖胞苷溶液添加到每皿5μM的终浓度逮捕神经胶质生长。 </li><li>通过在每个进给用2.5毫升的新鲜培养基更换旧培养基的2mL的订阅将细胞每周一次。加入额外的培养基是用于蒸发随时间进行补偿，并且只有介质的一部分被改变由神经胶质细胞，以确保介质的相一致调理。这些文化通常是对健康至少一个月（图3）。神经元的存活可通过加入APV到培养基中至100μM的最终浓度来提高。 APV是NMDA受体拮抗剂，并通过减少谷氨酸兴奋毒性促进存活。 </li></ol>

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作者什么都没有透露。

虽然培养神经元的"夹心层"的方法已在别处良好描述2， 如图11所示 ，在整个协议的几个步骤是相当困难的独自描述的文字，这可能会导致沮丧谁希望采纳调查。 该方法可分为三大工作流程:神经胶质培养，盖玻片制备和神经元培养物和维护。这三个准备的是高品质的神经元培养和几个重要的关键因素，必须牢记。电镀神经元前，神?...

大脑分为神经元的复杂网络。单个神经元到网络活动和脑功能的贡献可以通过它们的分子组成和它们的生理特性的扰动的选择性改变进行研究。单个神经元的遗传和化学处理是在培养的神经元比完整的脑组织受到后者的细胞的异质性和复杂性可以说是容易，无牵无挂。在培养神经细胞发展良好定义的轴突和树突乔木和彼此形成广泛的突触连接。 而来自成年动物或来自神经系统的其他区域神经元培养物是可能的，胚胎海马培养物经常由于优选为所定义的锥体细胞群体和相对低的密度胶质1,2。在培养低密度生长的海马神经元是特别AMEnable到亚细胞定位，蛋白质运输，神经元极性和突触发育的研究。在培养神经细胞也在突触可塑性3，4，5，6研究分子过程被广泛使用。从与不出生后存活的全球基因缺失的小鼠神经元文化的准备工作已经在研究某些基因7的细胞和突触的角色特别有用。 由于在脑，海马神经元依赖于从神经胶质细胞营养支持。这他们的文化复杂化，并导致由这种支持提供几种不同的方法的发展。一种常用的方法包括直接电镀到神经元的神经胶质细胞8，或允许从所获取的HIPP污染物神经胶质细胞的单层ocampal组织增殖并形成神经元9下方的单层。虽然这种方法已经发现了一些成功，得到的神经元培养物中的杂质是不利的成像实验。神经元培养物的另一种常用的方法是将离开时的胶质饲养层完全，而是提供在限定的生长培养基10的形式营养支持。 在这里，我们描述了"三明治"或神经元培养2,11的"银行家"的方法。此方法涉及镀覆在玻璃盖玻片上，然后将其悬浮在由石蜡珠分离的神经胶质细胞的单层的海马神经元。这有利于神经细胞的同质群体的长期培养，而不会污染的神经胶质细胞，同时允许从底层的神经胶质单层营养支持。当神经元是足够的年龄和成熟程度的，神经元盖玻片可翻转时的胶质菜和在成像或功能测定法中使用。

<table><tbody><tr><td>&lt;strong&gt;Dissection Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Micro Dissecting Scissors</td><td>Roboz</td><td>RS-5910</td><td></td></tr><tr><td>Micro Dissecting Spring Scissors</td><td>Roboz</td><td>RS-5650</td><td></td></tr><tr><td>Micro Dissecting Spring Scissors</td><td>Roboz</td><td>RS-5605</td><td></td></tr><tr><td>Dumont Forceps (#5)</td><td>Roboz</td><td>RS-5045</td><td></td></tr><tr><td>Dumont Forceps (#PP)</td><td>Roboz</td><td>RS-4950</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Tissue Preparation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Trypsin (2.5%)</td><td>Gibco</td><td>15-090-046</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%)</td><td>Gibco</td><td>25-200-072</td><td></td></tr><tr><td>Swinnex Filter Holder, 25&amp;nbsp;mm</td><td>EMD Millipore</td><td>SX0002500</td><td>Used as cell strainer. Assemble first with filter and autoclave</td></tr><tr><td>Isoflorane</td><td>Pharmaceutical Partners of Canada Inc.</td><td>CP0406V2</td><td></td></tr><tr><td>Hemocytometer</td><td>Hausser Scientific</td><td>1492</td><td></td></tr><tr><td>Grade 105 Lens Cleaning Tissue</td><td>GE Healthcare</td><td>2105-841</td><td>Used as cell strainer. Assemble first in filter holder and autoclave</td></tr><tr><td>Glass Pasteur pipettes with cotton filter</td><td>VWR</td><td>14672-412</td><td></td></tr><tr><td>HEPES (1 M)</td><td>Gibco</td><td>15-630-080</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution without Calcium, Magnesium, Phenol Red (HBSS, 10x)</td><td>Gibco</td><td>14-185-052</td><td></td></tr><tr><td>Glass Pasteur pipettes</td><td>VWR</td><td>14672-380</td><td></td></tr><tr><td>Deoxyribonuclease I from bovine pancreas (Dnase)</td><td>Sigma-Aldrich</td><td>DN25-100mg</td><td></td></tr><tr><td>Butane bunsen burner</td><td>Wall-Lenk Mfg. Co.</td><td>Model 65</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810R</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Tissue Culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Gibco</td><td>15-140-122</td><td></td></tr><tr><td>Petri Dish (100 mm)</td><td>Fisher</td><td>FB0875712</td><td></td></tr><tr><td>Petri Dish (60 mm)</td><td>Fisher</td><td>FB0875713A</td><td></td></tr><tr><td>Horse Serum</td><td>Gibco</td><td>16050-122</td><td>Can be used in place of BGS, but each lot must be tested due to inter-lot variation. Heat-inactivation of serum is recommended.</td></tr><tr><td>Sodium Hydroxide</td><td>Fisher Scientific</td><td>S318-1</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Sigma-Aldrich</td><td>P2256</td><td></td></tr><tr><td>Minimum Essential Medium (MEM)</td><td>Gibco</td><td>11-095-080</td><td></td></tr><tr><td>Neurobasal Medium</td><td>Gibco</td><td>21-103-049</td><td></td></tr><tr><td>L-Glutamine (200 mM)</td><td>Gibco</td><td>25-030-081</td><td></td></tr><tr><td>GlutaMAX Supplement</td><td>Gibco</td><td>35050061</td><td>Can be used in place of L-Glutamine in the NBG medium</td></tr><tr><td>Culture Dish (60 mm)</td><td>Corning, Inc</td><td>353002</td><td></td></tr><tr><td>Culture Flasks (75 cm^2)</td><td>Greiner Bio-One</td><td>658170</td><td></td></tr><tr><td>Cytarabine (Ara-C)</td><td>Sigma-Aldrich</td><td>C3350000</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td></td></tr><tr><td>Bovine Growth Serum (BGS)</td><td>HyClone</td><td>SH3054103</td><td>Heat-inactivation is recommended before use.</td></tr><tr><td>B27 Supplement (50x)</td><td>Gibco</td><td>17-504-044</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D8418-100ml</td><td></td></tr><tr><td>Cryogenic Vials</td><td>VWR</td><td>89094-806</td><td></td></tr><tr><td>DL-2-Amino-5-phosphonopentanoic acid (APV)</td><td>Sigma-Aldrich</td><td>A5282</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Coverslip Preparation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sodium tetraborate decahydrate (borax)</td><td>Sigma-Aldrich</td><td>B9876-1KG</td><td></td></tr><tr><td>Poly-L-Lysine Hydrobromide</td><td>Sigma-Aldrich</td><td>P2636</td><td></td></tr><tr><td>Histoplast Paraffin Wax</td><td>Fisher</td><td>22-900-700</td><td></td></tr><tr><td>Gravity Convection Oven</td><td>VWR</td><td>89511-404</td><td>Used for alternative coverslip cleaning method discussed in protocol</td></tr><tr><td>Ultrasonic Bath (Sonicator)</td><td>Fisher Scientific</td><td>15337400</td><td></td></tr><tr><td>Nitric Acid</td><td>Anachemia</td><td>62786-460</td><td></td></tr><tr><td>Ceramic Staining Racks</td><td>Thomas Scientific</td><td>8542E40</td><td>Used for alternative coverslip cleaning method discussed in protocol</td></tr><tr><td>Coverslips</td><td>Glaswarenfabrik Karl Hecht&amp;nbsp;GmbH</td><td>1001/18</td><td>Manufacturer is very important, as neurons do not adhere well to lower quality glass&amp;nbsp;</td></tr><tr><td>Boric Acid</td><td>Sigma-Aldrich</td><td>B0252</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Miscellaneous&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sterile Syringe Filters</td><td>VWR</td><td>28145-477</td><td>Used with BD syringe for filter-sterilization</td></tr><tr><td>Syringe&amp;nbsp;</td><td>BD</td><td>302832</td><td>Used with VWR sterile syringe filters for fliter-sterilization</td></tr><tr><td>Water Bath</td><td>Fisher Scientific</td><td>15-460-16Q</td><td></td></tr><tr><td>Inverted Microscope</td><td>Olympus</td><td>CKX41</td><td></td></tr><tr><td>15 mL Conical Sterile Centrifuge Tubes</td><td>ThermoScientific</td><td>339650</td><td></td></tr><tr><td>50 mL Conical Sterile Centrifuge Tubes</td><td>ThermoScientific</td><td>339652</td><td></td></tr></tbody></table>

low-density primary hippocampal neuron culture

探测单个神经细胞的培养物中的结构和生理的能力对于神经生物学的研究是至关重要的，并且允许在单个细胞或定义的网络的遗传和化学操作灵活性。相比于完整的脑组织时操纵这样便于在减少培养系统简单。尽管存在这些初级神经元的分离和生长方法很多，每个人都有其自身的局限性。这个协议描述了一种用于在玻璃盖玻片上，然后将其悬浮在神经胶质细胞的单层培养低密度和高纯度的啮齿动物胚胎海马神经元的方法。这种"三明治文化"允许同时允许从底层的神经胶质单层营养支持的神经元群的独家长期增长。当神经元是足够的年龄或到期水平的，神经元盖玻片可翻转时的胶质菜和在成像或功能测定法中使用。神经元摹rown通过该方法通常存活数周和发展广泛的乔木，突触连接，以及网络属性。

在初级神经细胞培养物的这种"三明治"方法，海马神经元（ 图3）上生长由石蜡珠（ 图2）中分离的神经胶质细胞（ 图1）的床层。这确保了神经元在玻璃盖玻片上选择性地生长以最小的神经胶质细胞污染，但接收来自胶质细胞上的组织培养皿生长足够营养支持。通常情况下，神经元可在培养基中维持&gt; 3周，开发具有发达的轴突和树?...

Watch this Scientific Journal Video about Low-Density Primary Hippocampal Neuron Culture at JoVE.com

Low-Density Primary Hippocampal Neuron Culture

本文介绍用于培养低密度初级海马神经元上倒置在神经胶质单层玻璃盖玻片生长的协议。神经元和神经胶质层通过石蜡珠分离。通过该方法生长的神经元适合于高分辨率光学成像和功能测定法。

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 ...

low-density-primary-hippocampal-neuron-culture

Research

JoVE Journal

Neuroscience

20.5K 次观看.  University of Manitoba. 本文介绍用于培养低密度初级海马神经元上倒置在神经胶质单层玻璃盖玻片生长的协议。神经元和神经胶质层通过石蜡珠分离。通过该方法生长的神经元适合于高分辨率光学成像和功能测定法。 

视频: Low-Density Primary Hippocampal Neuron Culture

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

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 ...

科研

神经科学

Primary Culture of Hippocampal Neurons from P0 Newborn Rats

The physiological properties of hippocampal neurons are commonly investigated, especially because of the involvement of the hippocampus in learning and memory. Primary hippocampal cell culturing allows neuroscientists to examine the activity and properties of neurons at the individual cell and single synapse level. In this video, we will demonstrate how to isolate and grow primary hippocampal cells from newborn rats. The hippocampus may be isolated from each newborn animal in as short as 2 to 3 minutes, and the cultures can be maintained for up to two weeks. We will also briefly demonstrate how to use these hippocampal neurons for ratiometric calcium imaging. While this protocol describes the process for the hippocampus, with little to no modification, it can be applied to other regions of the brain.

The dissection and growth of cells from an individual brain area facilitates investigation of cellular and physiological parameters. We describe a method for primary cell culturing that produces neuron-enriched cultures in a serum-free environment.

从P0新生大鼠海马神经元的原代培养

The physiological properties of hippocampal neurons are commonly investigated, especially because of the involvement of the hippocampus in learning and ...

生物学

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 ...

Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms responsible for those differences remain unclear. Primary disassociated embryonic neuronal culture provides a simplified experimental model with which to investigate the neuronal cell signaling involved in cell death as a result of ischemia or disease; however, most neuronal cultures used in research today are mixed sex. Researchers can and do test the effects of sex steroid treatment in mixed sex neuronal cultures in models of neuronal injury and disease, but accumulating evidence suggests that the female brain responds to androgens, estrogens, and progesterone differently than the male brain. Furthermore, neonate male and female rodents respond differently to ischemic injury, with males experiencing greater injury following cerebral ischemia than females. Thus, mixed sex neuronal cultures might obscure and confound the experimental results; important information might be missed. For this reason, the Herson Lab at the University of Colorado School of Medicine routinely prepares sex-stratified primary disassociated embryonic neuronal cultures from both hippocampus and cortex. Embryos are sexed before harvesting of brain tissue and male and female tissue are disassociated separately, plated separately, and maintained separately. Using this method, the Herson Lab has demonstrated a male-specific role for the ion channel TRPM2 in ischemic cell death. In this manuscript, we share and discuss our protocol for sexing embryonic mice and preparing sex-stratified hippocampal primary disassociated neuron cultures. This method can be adapted to prepare sex-stratified cortical cultures and the method for embryo sexing can be used in conjunction with other protocols for any study in which sex is thought to be an important determinant of outcome.

Primary disassociated embryonic hippocampal neuronal cultures are useful for investigating the signaling mechanisms involved in neuron death. Sexing the embryos before the isolation and dissociation of the hippocampus allows the preparation of separate male and female cultures, which enables the researcher to identify and investigate sex-specific cell signaling.

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms ...

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (&#916;E-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.

We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.

动物的基因分型快速其次是建立大脑神经元原代培养

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic hippocampal neurons can often grow successfully on glass coverslips at high density under serum-free conditions, but low density cultures typically require a supply of trophic factors by co-culturing them with a glia feeder layer, preparation of which can be time-consuming and laborious. In addition, the presence of glia may confound interpretation of results and preclude studies on neuron-specific mechanisms. Here, a simplified method is presented for ultra-low density (~2,000 neurons/cm2), long-term (&#62;3 months) primary hippocampal neuron culture that is under serum free conditions and without glia cell support. Low density neurons are grown on poly-D-lysine coated coverslips, and flipped on high density neurons grown in a 24-well plate. Instead of using paraffin dots to create a space between the two neuronal layers, the experimenters can simply etch the plastic bottom of the well, on which the high density neurons reside, to create a microspace conducive to low density neuron growth. The co-culture can be easily maintained for &#62;3 months without significant loss of low density neurons, thus facilitating the morphological and physiological study of these neurons. To illustrate this successful culture condition, data are provided to show profuse synapse formation in low density cells after prolonged culture. This co-culture system also facilitates the survival of sparse individual neurons grown in islands of poly-D-lysine substrates and thus the formation of autaptic connections.

Low density cultures of primary hippocampal neurons usually require glia feeder layer to supply neurotrophic factors and sustain longevity. We describe here a simplified method to culture ultra-low density neurons on glass coverslips in the presence of a high density neuronal feeder layer, which facilitates investigation of specific neuronal-autonomous mechanisms.

的简化方法超低密度，长期主要海马神经元文化

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic ...

Super-Resolution Imaging to Study Co-Localization of Proteins and Synaptic Markers in Primary Neurons

Synapses are the functional elements of neurons and their defects or losses are at the basis of several neurodegenerative and neurological disorders. Imaging studies are widely used to investigate their function and plasticity in physiological and pathological conditions. Because of their size and structure, localization studies of proteins require high-resolution imaging techniques. In this protocol, we describe a procedure to study in primary neurons the co-localization of target proteins with synaptic markers at a super-resolution level using structured illumination microscopy (SIM). SIM is a patterned-light illumination technique that doubles the spatial resolution of wide-field microscopy, reaching a detail of around 100 nm. The protocol indicates the required controls and settings for robust co-localization studies and an overview of the statistical methods to analyze the imaging data properly.

This protocol shows how to employ super-resolution microscopy to study protein co-localization in primary neuronal cultures.

超分辨率成像研究原发神经元中蛋白质和突触标记的共同定位

Synapses are the functional elements of neurons and their defects or losses are at the basis of several neurodegenerative and neurological disorders. ...

Easy and Reproducible Low-Density Primary Culture using Frozen Stock of Embryonic Hippocampal Neurons

Neuronal culture is a valuable system for evaluating synaptic functions and drug screenings. In particular, a low-density culture of primary hippocampal neurons allows the study of individual neurons or subcellular components. We have shown subcellular protein localization within a neuron by immunocytochemistry, neuronal polarity, synaptic morphology, and its developmental change using a low-density primary hippocampal culture. Recently, ready-to-use frozen stocks of neurons have become commercially available. These frozen stocks of neurons reduce the time needed to prepare animal experiments and also contribute to the reduction of the number of animals used. Here, we introduce a reproducible low-density primary culture method using a 96-well plate. We used a commercially available frozen stock of neurons from the rat embryonic hippocampus. The neurons can be stably cultured long-term without media changes by reducing the growth of glial cells at particular timepoints. This high-throughput assay using low-density culture allows reproducible imaging-based evaluations of synaptic plasticity.

A ready-to-use frozen stock of neurons is a powerful tool for evaluating synaptic functions. Here, we introduce an easy low-density primary culture from frozen stock using a 96-well plate.

使用胚胎海马神经元冷冻原液进行简单且可重复的低密度原代培养

Neuronal culture is a valuable system for evaluating synaptic functions and drug screenings. In particular, a low-density culture of primary hippocampal ...

A Technique to Culture Mouse Hippocampal Neurons

Source: Yang, R., et al. <a href="https://app.jove.com/v/61411">Use of Primary Cultured Hippocampal Neurons to Study the Assembly of Axon Initial Segments.</a> J. Vis. Exp. (2021)
The video demonstrates a method for culturing neurons from murine hippocampi. Initially, the hippocampi undergo enzymatic digestion to break down the extracellular tissue matrix. Subsequently, the digested tissue is mechanically disrupted to isolate individual neurons. Finally, these neurons are cultured on glial feeder cells.

一种培养小鼠海马神经元的技术

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparing glia cell feeder dishes ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Primary Neuronal Culture

Generating a Low-Density Primary Neuron Culture From Frozen Embryonic Neurons

Source: Koganezawa, N. et al., <a href="https://app.jove.com/v/64872">Easy and Reproducible Low-Density Primary Culture using Frozen Stock of Embryonic Hippocampal Neurons.</a>&#160;J. Vis. Exp. (2023)
This video demonstrates the method of culturing cryopreserved embryonic neurons, involving controlled thawing, dilution with a neurobasal medium, and seeding onto a poly-L-lysine-coated plate. Subsequent incubation supports the growth and maintenance of neuronal connections.

从冷冻胚胎神经元生成低密度原代神经元培养物

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

	Coat a 96-well ...