The authors thank researchers at the University of Iowa, Drs. Luis Tecedor, Ines Martins and Beverly Davidson for instructions and helpful comments regarding striatal cultures, and Drs. Kara Gordon, Nicole Bode and Pedro Gonzalez-Alegre for genotyping assistance and discussions. We also thank Dr. Eric Weyand (Animal Identification and Marking Systems) for helpful comments regarding tattooing, and Dr. Shutaro Katsurabayashi (Fukuoka University) for helpful comments regarding the mouse culture. This work was supported by grants from the American Heart Association, the Department of Defense (Peer Reviewed Medical Research Program award W81XWH-14-1-0301), the Dystonia Medical Research Foundation, the Edward Mallinckrodt, Jr. Foundation, the National Science Foundation, and the Whitehall Foundation (N.C.H.).

注:在本研究中进行的所有动物的程序批准了美国爱荷华大学的机构动物护理和使用委员会。 利用小鼠的纹身爪垫1.长期鉴定<ol><li>固定用爪子垫（足底面）一爪子对着实验者。持用拇指和食指的爪子。要小心，不要捏爪子。 注:稳定的固定化是很重要的，以确保该纹身颜料放入爪垫的真皮，并因此是永久性7。 </li><li>拭爪垫，用70％的乙醇在纱布海绵或拭子。 </li><li>涂抹护肤油以一个棉签，轻轻按压脚掌垫几次面的提示。只使用少量皮肤油;当存在时大量，它将阻止纹身颜料到达皮肤。 </li><li>浸纹身针的顶端到纹身油墨之前以纹身。使用清洁的，无菌和锋利纹身针，以减少疼痛和感染的可能性，并且还增加了纹身效率。 </li><li>而爪垫表面覆盖有皮肤油，垂直和轻轻按纹身针尖对在爪垫的中心的皮肤，和多次喷射墨。见表材料/设备关于电动纹身系统的信息。 </li><li>喷70％乙醇的纱布海绵，轻轻按压在足去除多余的纹身色素在皮肤表面，并检查纹身的质量。检查爪垫中间有一个黑暗，圆斑不同于正常皮肤色素沉着。如果纹身是没有黑暗，或方便查看足够大，重复之前的步骤纹身。 </li></ol> 2.使用快速PCR基因分型基因分型试剂盒新生小鼠<ol><li>消毒的小鼠尾巴用70％乙醇的远端，切割5mm或更小的尾部倾斜，并将其传输到用于PCR的类型的8条管的管。使用未使用刀片的每个小狗避免样本之间的交叉污染。或者，使用剪刀，但是在这种情况下，小心地和彻底地除去剩余的组织上用70％的乙醇中的刀片。检查出血。如果发生出血，施加压力的尾部用纱布海绵切割部分直到出血停止。 </li><li>添加200μl的DNA提取溶液到含有样品每个PCR管中。见表材料/设备及其有关的工具包组成和信息。 </li><li>将管条置于PCR热循环仪，并且使用下面的程序开始了DNA提取:1个循环的55℃10分钟，1个循环，在95℃进行10分钟，并保持在4℃。 注意:这是在稍后用于PCR相同的PCR热循环仪。 </li><li>经过DNA提取完成后，从热循环仪取出一管带次颠倒5次。 </li><li>转印各试样的4微升溶液（DNA提取物），以未使用的8管带的管，和与混合:10微升2X PCR预拌二，2微升混合正向＆反向引物（建议在0.5μM; 见表材料/设备的序列），和4微升不含核酸酶的H 2 O为每一个试样。使用台式离心机旋短暂（ 例如，3秒）。置于冰上在任何时候都管处理时除外。 </li><li>进行热循环。见 表材料/设备的热程序。 </li><li>检测扩增的DNA产物。装载从PCR（20微升）的总反应溶液直接进琼脂糖凝胶的孔中。加载分子量标记到一个单独的井。施加电场。 </li><li>取得频带的荧光图像在紫外光下。 </li></ol>鼠脑神经元3.原代培养S上胶质滋养层注意:程序脑解剖和细胞解离（3.1）是共同的所有后续步骤。对于小鼠胶质细胞培养的过程（3.2），大鼠神经胶质培养物（3.3），和小鼠的神经元培养物（3.4）分别描述之后。 <ol><li>脑解剖和细胞Dissocaiation <ol><li>牺牲一只小鼠或大鼠幼崽断头，迅速 ​​取出大脑，并放置到Hanks&#39;液（ 见表1对组合物）+ 20％在35毫米的培养皿的胎牛血清（FBS）（保持在冰上）。 </li><li>划破中线大脑分成两个半球。从大脑的每个半球除去感兴趣的区域（ 例如，大脑皮质，纹状体和海马）。从表面去除脑膜和主要血管。切开大脑区域到使用手术刀片4-10薄片。 </li><li>冲洗脑切片在一个15毫升的离心管中，一次机智ħHanks液+ 20％FBS中，然后3次，用Hanks液（ 即无血清）（所有4℃）。冲洗，加入5-10毫升溶液，让大脑切片沉降在管的底部，抽吸从顶部的溶液，并加入新鲜的溶液中。通过抽吸液完成清洗过程。 </li><li>滤波器2毫升含胰蛋白酶消化溶液（ 见表1对组合物）使用3毫升注射器和一个0.2微米的注射器过滤器，和直接添加滤液到含有脑片管。让胰蛋白酶消化进行在室温13分钟。 </li><li>通过吸大部分，然后加入7-10毫升Hanks液+ 20％FBS（4℃）的第一中和胰蛋白酶溶液。 </li><li>用Hanks冲洗脑切片，两次&#39;溶液+ 20％FBS中，然后三次用Hanks液（ 即无血清）（所有4℃）。通过吸取SOLUT完成漂洗程序离子。 </li><li>过滤2毫升解离溶液（ 见表1对组合物）使用3毫升注射器和一个0.2微米的注射器过滤器，和直接添加滤液到管与脑切片。 </li><li>机械轻轻研制过程10-20次，直到可见组织块消失分离的细胞。用棉花塞住，火抛光的巴斯德吸管，避免研磨过程中制作气泡。 </li><li>等待3分钟的小片安定下来。 </li><li>传送大部分的溶液（〜1.5毫升，留下一些溶液在底部）到包含3毫升Hanks液+ 20％FBS中的溶液（4℃）一个15毫升的离心管中，用棉花塞住，拒爆─抛光的巴斯德吸管。不转移的所有解决方案，因为包含任何沉积物的底部典型地导致在培养的劣化。 </li><li>离心13分钟到185克（〜1100转），在4℃。 </li><li>吸出上清液轻轻地，加入1毫升预热的电镀液（37℃），以沉淀，并轻轻吹打使用棉栓，火抛光的巴斯德吸管数次重悬它。 注:三种不同类型的电镀介质用于不同培养用途:镀介质-1小鼠的神经胶质细胞，镀介质-2小鼠的神经元，和电镀介质-3鼠神经元和神经胶质细胞（ 见表1的组合物） 。 </li><li>取出10微升该细胞悬浮液，用10微升的0.4％台盼蓝溶液混合，并测量活细胞密度，使用一个血球或自动细胞计数器。 </li></ol></li><li>鼠标胶质细胞培养<ol><li>洗盖玻片，以帮助建立小鼠细胞的健康文化。 <ol><li>沉浸盖玻片（圆形，直径12mm）在70％硝酸中的玻璃培养皿。使用铝箔避光，并将其放置在轨道摇床O / N。 </li><li>冲洗玻璃coversliPS在培养皿至少三次蒸馏水。沉浸其中在蒸馏水中。放在轨道摇床O / N的菜。 </li><li>干上了生物安全柜用Whatman150毫米滤纸盖玻片。 </li><li>高压灭菌盖玻片。 </li><li>放置盖玻片入24孔培养皿。 </li></ol></li><li>按照步骤1和2标签和基因型新生小鼠。 </li><li>获得来自小鼠幼仔的脑细胞，根据步骤3.1。 注:使用大脑皮层的是典型的准备鼠标胶质滋养层。然而，其他的大脑区域，例如海马和纹状体，将细胞密度的适当调整后的工作，由于不同的号码，并从不同的区域的细胞的产率。 </li><li>添加〜4毫升预热镀介质-1至最终细胞悬浮液（约1毫升）中。对于预热的培养基，放置在培养箱中的溶液（5％CO 2 -95％O 2，37℃）O / N，以允许的温度和pH值下保持稳定。 </li><li>细胞悬液转移到一个未涂覆的T25培养瓶中，用棉花塞住，火抛光的巴斯德吸移管。放置在所述培养箱中的烧瓶中。 </li><li>在第1天在体外 （DIV），冲洗培养的细胞在T25烧瓶中以电镀两次培养基-1（4℃）。将烧瓶放回培养箱。执行由抽吸烧瓶完全用巴氏吸管内侧的介质，加入约5毫升的新鲜培养基，并轻轻倾斜烧瓶几次以旋涡运动漂洗。 </li><li>在6-9 DIV，（ 即，一天胰蛋白酶消化前和电镀盖玻片上，在步骤3.2.8-3.2.13），放置100微升涂层材料与细胞外基质蛋白（见表材料/设备有关的评论涂层材料）上的盖玻片在培养皿，然后将培养皿在培养箱中。 </li><li>在7-10 DIV，当细胞是20-40％汇合（空间连续），trypsinize它们。 <ol><li>冲洗烧瓶一次，通过吸烧瓶内的所有的溶液并加入〜13毫升的Hanks&#39;液（4℃），轻轻倾斜烧瓶多次在一个旋涡运动，并完全吸出溶液。 </li><li>加入40微升DNA酶溶液（终浓度，750单位/ ml）到4ml胰蛋白酶-EDTA溶液，通过0.2μm的注射器过滤该溶液，并直接添加滤液至细胞在烧瓶中。 </li><li>让胰蛋白酶消化进行13分钟，在37℃的培养箱中培养。 </li><li>加入2毫升100％FBS（4℃）至烧瓶中和胰蛋白酶溶液。 </li><li>传送胰蛋白酶细胞用5至10毫升吸管一个15毫升的离心管中，加入Hank氏溶液+ 20％〜4毫升FBS的（4℃），离心〜185克和4℃下进行13分钟，并吸出上清液。 </li></ol></li><li>重悬沉淀在1ml预WÄRME的ð电镀介质-1。 </li><li>根据步骤3.1.13测量细胞的密度。 </li><li>吸出涂覆材料完全从玻璃盖玻片，并且板〜50微升的重新悬浮的神经胶质细胞的盖玻片上。 注:这些细胞将建立胶质滋养层。 </li><li>放置在培养皿中孵化盖玻片。 </li><li> 20-60分钟后，加入1 ml预热的镀介质-1向每个孔中，然后将培养皿放回培养箱中。注意:当镀介质被加入，这将是有帮助的，以使用另一个吸管压下盖玻片（在没有镀细胞）的外周，从而使盖玻片不会在培养基中漂浮。 </li><li>在1 DIV胶质饲养层（ 即，在玻璃盖玻片）的，用1ml更换介质预热镀介质-1，由抽吸所有的孔中的溶液中，然后用新鲜的培养基填充它。 </li><li>在2-3 DIV胶质饲养层的CEL时LS是80-100％汇合时，加有丝分裂抑制剂（10μl的5-氟-2&#39;-脱氧尿苷+尿苷的混合物;终浓度为81.2和204.8μM，分别地）到每个孔中，以抑制DNA复制，因此，以抑制神经胶质细胞的增殖。 </li><li>在7-9 DIV，当细胞是90-100％汇合（胶质饲养层上镀敷的神经元前1-2小时），以取代所述培养基预热镀介质-2（37℃）。 注:神经元将被镀在同一天，使用步骤3.4。 </li></ol></li><li>神经胶质文化<ol><li>涂覆T25培养瓶用相同的涂层材料。 注意:这是必要的，在步骤3.3.5以后除去非粘附细胞。 <ol><li>加入2.0毫升涂层材料至该烧瓶中，并将其放置在所述培养箱中的烧瓶中。 </li><li>后2-3小时，完全吸出涂​​覆材料，使得烧瓶中的地板变干燥。 </li></ol></li><li>获取细胞从大鼠幼仔，根据步骤3.1。 注:海马的CA3-CA1区用于制备大鼠胶质饲养层。然而，其他的大脑区域，例如大脑皮质和纹状体，将调整为在细胞密度由于不同数量和不同区域的细胞的产率的差异后工作。 </li><li>添加〜4毫升预热镀介质-3至最终细胞悬浮液（约1毫升）中。 </li><li>细胞悬液转移到涂覆的T25培养瓶中，用棉花塞住，火抛光的巴斯德吸移管。将烧瓶中的培养恒温箱（5％CO 2 -95％O 2，37℃）。 </li><li>在2-3 DIV，当细胞是20-40％汇合时，除去非粘附或弱粘附细胞，通过关闭盖子紧紧，摇动烧瓶大力〜10倍，抽吸该溶液，并加入4-5镀毫升介质-3（4℃）。重复此步骤一次。检查整个烧瓶地板的培养细胞（ 例如，用相差显微镜），以确认那些出现神经元（ 即那些的量，电池主体的外边缘出现相亮）被完全除去。根据需要重复该过程经常以除去这些细胞，然后将烧瓶返回到培养箱中。 注:实力和"奶昔"要求可能会有所不同个体之间的实验者的总数。重要的是不保持奶昔的总数的一组数字，但证实神经元样细胞被消除。 </li><li>在6-8 DIV，当细胞是90-100％汇合时，通道中的神经胶质细胞通过胰蛋白酶它们​​作为在步骤3.2.8。 </li><li>离心后，重悬沉淀在1ml电镀介质-3（4℃）中，加10镀介质-3（4℃）中的溶液中，胰蛋白酶消化细胞转移到未涂覆的T75烧瓶中，并将其培养在孵化器。 注:鼠的神经胶质细胞将传代两次;与此相反的小鼠的神经胶质细胞ARË代只有一次，因为他们成为多次传代后，不健康的。大鼠神经胶质细胞将传代以未涂覆的任何涂层材料T75烧瓶。该涂层允许大鼠神经胶质细胞生长太快而产生许多纤毛细胞（推定的室管膜细胞），这将在后面恶化的神经元的培养条件。 </li><li>在烧瓶中17-19 DIV胶质培养物（ 即，传代和1天胰蛋白酶消化前和通过步骤3.3.9-3.3.14镀到盖玻片后11天），将100微升的涂覆材料上未洗涤盖玻片（圆形，直径12mm）在培养皿中，然后将培养皿在培养箱中。 注:对于神经胶质文化的差异并没有注意到，在培养结果有或没有清洗的盖玻片。 </li><li>在烧瓶18-20 DIV胶质文化（ 即传代12天后），通过胰蛋白酶培养细胞准备胶质滋养层，根据STEP 3.2.8。 </li><li>在离心过程中，从玻璃盖玻片完全吸出涂​​料。 </li><li>离心后，重悬沉淀在1ml电镀介质-3（4℃）的，并测量细胞密度，根据步骤3.1.13。 </li><li>调整胶质密度至10 4活细胞/ ml，和板100微升胶质细胞悬浮在涂覆的玻璃盖玻片上。 注:这些细胞将建立胶质滋养层。 </li><li>放置培养皿用盖玻片放入培养箱为20-60分钟。 </li><li>加入1毫升镀介质-3（4℃）至各孔中，然后将培养皿放回培养箱中。 </li><li>在3-4 DIV胶质饲养层，当在盖玻片细胞是40-80％汇合时，加入1毫升预热的生长培养基（ 见表1对组合物），包含胞嘧啶β-D-阿拉伯呋喃糖苷（阿糖胞苷，4μM终浓度）来停止神经胶质细胞的增殖。板块神经s的7-9 DIV胶质饲养层时，细胞是60-80％汇合时，使用步骤3.4。 </li></ol></li><li>鼠标神经细胞<ol><li>按照步骤1和2标签和基因型新生小鼠。 </li><li>使用鼠标幼崽步3.1。 </li><li>调整的活细胞悬浮液的密度，以〜2.0×10 5细胞/ ml的用预温热镀介质-2-电镀对小鼠的神经胶质细胞，或使用电镀介质-3-电镀对大鼠神经胶质细胞。 </li><li>板中的细胞中的胶质饲养层上，通过轻轻地加入细胞悬浮液到每个培养基良好。注:细胞悬浮液中添加的量将通过细胞的目标数在培养井来确定。例如，板〜60微升细胞悬浮液以达到12000个细胞/孔。 </li><li>需要注意的是无解的变化是必要的神经元​​镀后。要小心，以避免从孔中的溶液蒸发超过文化的过程中，通过加湿里面Øf显示培养孵化器。 </li></ol></li></ol>

The author (Zhengmin Huang) is the president of EZ BioResearch LLC that produces reagents described in this article.

这里介绍的协议包括程序纹身标记/识别小鼠的基因分型，从尾部的提示小鼠，并为培养小鼠大脑的神经元密度低。在一个循环中使用6-8幼仔的实验中，这些过程通常需要约0.5小时，〜4小时和〜2小时，分别在共6-7小时的。这使得实际用于单个实验者完成所有从幼仔的出生到神经元培养物的电镀时所必需的程序 - 在少于单个工作日（除胶质饲养层之前制备的）。 纹身 </p...

遗传疾病的啮齿动物模型已被证明在建立正常蛋白质和核酸，以及在这些缺陷的病理生理后果的生理功能非常有用。举出缺陷型为参与关键的细胞功能的蛋白质的小鼠，以及病症如阿耳茨海默氏病的小鼠模型。然而，某些遗传操作可导致新生儿杀伤力不久或在出生后几天。在这些情况下，原代细胞培养物，因为活细胞可以从死亡前的胚胎或新生儿幼仔得到的重要工具，它们可以保持在至少几个星期的体外 ，并在这段时间内的早期神经元发育，可接着生化，功能和形态的实验。对于主要培养物，也可以是有益的直接制版的神经元密度低;这使得可以以可视化的个别胞体，树突，轴突轴和神经TERMINALS在高的空间分辨率。然而，神经元在低浓度的存活和分化通常要求它们被镀上一个胶质饲养层，共培养的神经胶质细胞在不存在与它们由胶质1空调物理接触，或在培养基中培养。 低密度的神经元培养物的对神经胶质饲养层的机构可以是依赖于快速和可靠的基因分型预先 - 在几个小时在对比几天。速度是特别重要的神经基因型需要被匹配到预先制备的胶质饲养层时。作为更实际的例子，它可能有必要决定哪些其中基因型的幼仔在产生培养物的使用，以优化实验的效率。 在这里，我们证明了工作的协议，已被用于快速，简单和可靠的小鼠基因分型在以前的出版物2-6。鼠标的尾巴，市售的试剂盒的使用。这个协议包括从组织单步提取核酸，并且既不需要一个核酸纯化步骤，也不使用终止缓冲液（"停止溶液"）。这种基因分型方法的可靠性是通过提出的一系列试验的结果时，差异被相对于该样品的起始量，动物的年龄和PCR扩增子的长度引入的说明。该套件提供了自动提取和可靠性的优势。 为求是全面，使用纹身长期鉴定基因型小鼠的也证实。纹身是通过将纹身油墨对皮肤的真皮（表皮下）实现7。的过程被描述为纹身新生儿或1日龄小鼠的爪垫，虽然纹身可以应用到身体的其他部位，如尾和脚趾，并以动画所有年龄段的ALS。另外，程序将被证明为在低密度电镀和培养小鼠的神经元，是根据不同类型的神经胶质饲养层2,8的优化制剂。 我们使用继承神经系统疾病DYT1肌张力障碍的遗传小鼠模型-造成的基因TOR1A突变为常染色体显性运动障碍（c.904_906delGAG / c.907_909delGAG; p.Glu302del / p.Glu303del）9。所编码的蛋白质，torsinA，属于（AAA +）蛋白质家族，其成员通常执行伴侣样的功能，帮助在"与不同的细胞活性相关的ATP酶":蛋白质折叠，蛋白质复合物的拆卸，膜运输，和囊泡融合10-13。该突变导致一个在框内缺失谷氨酸的密码子的，并可能导致"早发性全身性肌张力障碍分离的"14,15的表现。但是，路径负责这种疾病ophysiological机制仍然知之甚少。在一个敲入小鼠模型中，该突变体等位基因是TOR1A tm2Wtd，以下简称TOR1AΔE提及。杂ΔE-torsinA基因敲除小鼠是可行和基因模仿人类患者的肌张力障碍DYT1，而纯合子基因敲除小鼠出生后16,17死，与延迟到出生后死亡受遗传背景的18。纯合敲除小鼠的早期死亡的必要条件是动物两者的基因分型，并建立的神经元培养物的迅速完成。作为分型的另一个例子，Tfap2a（转录因子AP-2α，活化增强子结合蛋白2α）将被使用。由该基因编码的蛋白质是在调节多种细胞过程，诸如增殖，分化，存活和凋亡19重要。

<table><tbody><tr><td>REAGENTS - tattooing</td><td></td><td></td><td></td></tr><tr><td>Machine Cleanser</td><td>Animal Identification and Marking Systems, Inc.</td><td>NMCR3</td><td>This is used to clean the needles and the holder after tattooing.</td></tr><tr><td>Machine Drying Agent</td><td>Animal Identification and Marking Systems, Inc.</td><td>NDAR4</td><td>This is used to dry the needles and holder after cleaning.</td></tr><tr><td>Neonate Tattoo Black Pigment</td><td>Animal Identification and Marking Systems, Inc.</td><td>NBP01</td><td></td></tr><tr><td>Skin Prep Applicator</td><td>Animal Identification and Marking Systems, Inc.</td><td>NSPA1</td><td>Q-tip.</td></tr><tr><td>Skin Prep solution</td><td>Animal Identification and Marking Systems, Inc.</td><td>NSP01</td><td>This reagent delivers a thin layer of oil that enhances the efficiency of tattooing and prevents tattoo fading, by (information from vendor): 1) preventing non-tattooed skin from being stained temporarily, thereby allowing the quality of a paw pad tattoo to be easily evaluated before the pup is returned to its home cage &amp;ndash; the stained skin surface can be confused with the tattooed skin, 2) reducing skin damage during tattooing &amp;ndash; softening the skin and lubricating the needle will help the needle penetrate the skin without causing skin damage, and 3) preventing molecular oxygen from entering the skin, thereby reducing inflammatory responses to reactive oxygen species that can be generated.</td></tr><tr><td>REAGENTS - genotyping</td><td></td><td></td><td></td></tr><tr><td>EZ Fast Tissue/Tail PCR Genotyping Kit (Strip Tube Format)</td><td>EZ BioResearch LLC</td><td>G2001-100</td><td></td></tr><tr><td>2X PCR Ready Mix II</td><td>EZ BioResearch LLC</td><td>G2001-100</td><td>A red, loading dye for electrophoresis is included in the 2X PCR Ready Mix solution.</td></tr><tr><td>Tissue Lysis Solution A</td><td>EZ BioResearch LLC</td><td>G2001-100</td><td>Prepare DNA Extraction Solution by mixing 20 &amp;micro;l of Tissue Lysis Solution A and 180 &amp;micro;l of Tissue Lysis Solution B per specimen.</td></tr><tr><td>Tissue Lysis Solution B</td><td>EZ BioResearch LLC</td><td>G2001-100</td><td>Prepare DNA Extraction Solution by mixing 20 &amp;micro;l of Tissue Lysis Solution A and 180 &amp;micro;l of Tissue Lysis Solution B per specimen.</td></tr><tr><td>Acetic acid, glacial</td><td>VWR</td><td>BDH 3092</td><td></td></tr><tr><td>Agarose optimized grade, molecular biology grade</td><td>rpi</td><td>A20090-500&amp;nbsp;</td><td>We use 2% agarose gels in TAE buffer containing the SYBR Safe DNA gel stain (diluted 10,000-fold) or ethidium bromide (0.5 &amp;micro;g/ml gel volume).</td></tr><tr><td>Ethidium bromide</td><td>Sigma-Aldrich</td><td>E7637-1G</td><td></td></tr><tr><td>Ethylenediamine tetraacetic acid, disodium salt dihydrate (EDTA)</td><td>Fisher</td><td>BP120-500</td><td></td></tr><tr><td>Filtered Pipet Tips, Aerosol-Free, 0.1-10 &amp;micro;l</td><td>Dot Scientific Inc</td><td>UG104-96RS&amp;nbsp;</td><td>Use pipette tips that are sterile and free of DNA, RNase and DNase. For all steps involving DNA, use filtered pipette tips to avoid cross-contamination.</td></tr><tr><td>Filtered Pipet Tips, Premium Fit Filter Tips, 0.5-20 &amp;micro;l</td><td>Dot Scientific Inc</td><td>UG2020-RS</td><td>Use pipette tips that are sterile and free of DNA, RNase and DNase. For all steps involving DNA, use filtered pipette tips to avoid cross-contamination.</td></tr><tr><td>Filtered Pipet Tips, Premium Fit Filter Tips, 1-200 &amp;micro;l</td><td>Dot Scientific Inc</td><td>UG2812-RS</td><td>Use pipette tips that are sterile and free of DNA, RNase and DNase. For all steps involving DNA, use filtered pipette tips to avoid cross-contamination.</td></tr><tr><td>Molecular weight marker, EZ DNA Even Ladders 100 bp</td><td>EZ BioResearch LLC</td><td>L1001</td><td>We use either of these three molecular weight markers.</td></tr><tr><td>Molecular weight marker, EZ DNA Even Ladders 1000 bp</td><td>EZ BioResearch LLC</td><td>L1010</td><td></td></tr><tr><td>Molecular weight marker, TrackIt, 100 bp DNA Ladder</td><td>GIBCO-Invitrogen</td><td>10488-058</td><td></td></tr><tr><td>PCR tubes, 8-tube strips with individually attached dome top caps, natural, 0.2 ml&amp;nbsp;</td><td>USA Scientific</td><td>1402-2900</td><td>Use tubes that are sterile and free of DNA, RNase and DNase. An 8-tube strip is easy to handle and to group the specimens than individual tubes.</td></tr><tr><td>PCR tubes, Ultraflux Individual&amp;nbsp;</td><td>rpi</td><td>145660</td><td>Use tubes that are sterile and free of DNA, RNase and DNase.</td></tr><tr><td>Seal-Rite 0.5 ml microcentrifuge tube, natural</td><td>USA Scientific</td><td>1605-0000</td><td>Use tubes that are sterile and free of DNA, RNase and DNase.</td></tr><tr><td>SYBR Safe DNA gel stain * 10,000x concentration in DMSO</td><td>GIBCO-Invitrogen</td><td>S33102</td><td></td></tr><tr><td>Tris base</td><td>rpi</td><td>T60040-1000</td><td></td></tr><tr><td>Primers for amplifying Tor1a gene in &amp;Delta;E-torsinA knock-in mice</td><td></td><td></td><td>5&#039;-AGT CTG TGG CTG GCT CTC CC-3&#039; (forward) and 5&#039;-CCT CAG GCT GCT CAC AAC CAC-3&#039; (reverse) (reference 18). These primers were used at a final concentration of 1.0 ng/&amp;micro;l (~0.16 &amp;micro;M) (reference 2).</td></tr><tr><td>Primers for amplifying Tfap2a gene in wild-type mice</td><td></td><td></td><td>5&#039;-GAA AGG TGT AGG CAG AAG TTT GTC AGG GC-3&#039; (forward), 5&#039;-CGT GTG GCT GTT GGG GTT GTT GCT GAG GTA-3&#039; (reverse) for the 498-bp amplicon, 5&#039;-CAC CCT ATC AGG GGA GGA CAA CTT TCG-3&#039; (forward), 5&#039;-AGA CAC TCG GGC TTT GGA GAT CAT TC-3&#039; (reverse) for the 983-bp amplicon, and 5&#039;-CAC CCT ATC AGG GGA GGA CAA CTT TCG-3&#039; (forward), 5&#039;-ACA GTG TAG TAA GGC AAA GCA AGG AG-3&#039; (reverse) for the 1990-bp amplicon. These primers are used at 0.5 &amp;micro;M.</td></tr><tr><td>REAGENTS - cell culture</td><td></td><td></td><td></td></tr><tr><td>5-Fluoro-2&amp;prime;-deoxyuridine</td><td>Sigma-Aldrich</td><td>F0503-100MG</td><td>See comments section of uridine for more information.</td></tr><tr><td>B-27 supplement</td><td>GIBCO-Invitrogen</td><td>17504-044</td><td></td></tr><tr><td>Cell Culture Dishes 35 x 10 mm Dishes, Tissue Culture-treated</td><td>BD falcon</td><td>353001</td><td></td></tr><tr><td>Cell Culture Flasks, T25, Tissue Culture-treated, Canted-neck, plug-seal cap, 25 cm&lt;sup&gt;2&lt;/sup&gt; Growth Area, 70 ml</td><td>BD falcon</td><td>353082</td><td></td></tr><tr><td>Cell Culture Flasks, T75, Tissue Culture-treated, Canted-neck, vented cap, 75 cm&lt;sup&gt;2&lt;/sup&gt; Growth Area, 250 ml</td><td>BD falcon</td><td>353136</td><td></td></tr><tr><td>Conical Tube, polypropylene, 15 ml</td><td>BD falcon</td><td>352095</td><td></td></tr><tr><td>Countess (cell number counter) chamber slides</td><td>GIBCO-Invitrogen</td><td>C10312</td><td></td></tr><tr><td>Cytosine &amp;beta;-D-Arabinofuranoside hydrochloride (Ara-C hydrochloride)</td><td>Sigma-Aldrich</td><td>C6645-100mg</td><td></td></tr><tr><td>D-(+)-Glucose (Dextrose) anhydrous, SigmaUltra, 99.5% (GC)</td><td>Sigma-Aldrich</td><td>G7528-250G</td><td></td></tr><tr><td>Dish, Petri glass 100 x 15 mm</td><td>Pyrex</td><td>3160-101</td><td></td></tr><tr><td>Distilled water</td><td>GIBCO-Invitrogen</td><td>15230-147</td><td></td></tr><tr><td>DNase Type II</td><td>Sigma-Aldrich</td><td>D4527-200KU</td><td>Stock solution is prepared at 1,500 units/20 &amp;mu;l = 75,000 units/ml in distilled water.</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM), high glucose, GlutaMAX, pyruvate</td><td>GIBCO-Invitrogen</td><td>10569-010, 500 ml</td><td></td></tr><tr><td>Fast PES Filter Unit, 250 ml, 50 mm diameter membrane, 0.2 &amp;micro;m Pore Size</td><td>Nalgene</td><td>568-0020</td><td></td></tr><tr><td>Fast PES Filter Unit, 500 ml, 90 mm diameter membrane, 0.2 &amp;micro;m Pore Size</td><td>Nalgene</td><td>569-0020</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>GIBCO-Invitrogen</td><td>26140-079</td><td></td></tr><tr><td>Glass coverslip, 12 mm Round, thickness 0.09&amp;ndash;0.12 mm, No. 0</td><td>Carolina</td><td>633017</td><td></td></tr><tr><td>GlutaMAX-I</td><td>GIBCO-Invitrogen</td><td>35050-061</td><td></td></tr><tr><td>Hanks&#039; Balanced Salts</td><td>Sigma-Aldrich</td><td>H2387-10X</td><td></td></tr><tr><td>HEPES, &amp;ge;99.5% (titration)</td><td>Sigma-Aldrich</td><td>H3375-250G</td><td></td></tr><tr><td>Hydrochloric acid, 37%, A.C.S reagent</td><td>Sigma-Aldrich</td><td>258148-100 ML</td><td></td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>I5500-250 mg</td><td></td></tr><tr><td>Magnesium sulfate heptahydrate, MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;(7H&lt;sub&gt;2&lt;/sub&gt;O), BioUltra, &amp;ge;99.5% (Fluka)</td><td>Sigma-Aldrich</td><td>63138-250G</td><td></td></tr><tr><td>Matrigel Basement Membrane Matrix solution, Phenol Red-Free</td><td>BD Biosciences</td><td>356237</td><td>This is the coating material for coverslips and flasks. 1) To prepare it, thaw the Matrigel Basement Membrane Matrix solution on ice, which usually takes ~1 day. Using a pre-cooled pipette, aliquot the thawed solution into pre-cooled T25 flasks on ice, and store the flasks at -20 &amp;deg;C. To prepare the working Matrigel solution, thaw the aliquotted Matrigel in a flask on ice, dilute 50-fold by adding pre-cooled MEM solution and keep the diluted solution at 4 &amp;deg;C. It is important to pre-cool all cultureware and media that come into contact with Matrigel, except during and after the coating of coverslips, to prevent it from prematurely forming a gel. 2) To coat the glass coverslips or culture flasks with Matrigel, apply the Matrigel solution to the surface. Before plating cells, it is important to completely dry up the surface. For this purpose, it might be helpful to aspirate Matrigel during the cellular centrifugation immediately before plating the cells and to allow enough time for drying.</td></tr><tr><td>Minimum Essential Medium (MEM)</td><td>GIBCO-Invitrogen</td><td>51200-038</td><td></td></tr><tr><td>MITO+ Serum Extender, 5 ml</td><td>BD Biosciences</td><td>355006</td><td></td></tr><tr><td>Multiwell Plates, Tissue Culture-treated 24-well plate</td><td>BD falcon</td><td>353047</td><td></td></tr><tr><td>Multiwell Plates, Tissue Culture-treated 6-well plate</td><td>BD falcon</td><td>353046</td><td></td></tr><tr><td>Neurobasal-A Medium (1X), liquid</td><td>GIBCO-Invitrogen</td><td>10888-022</td><td></td></tr><tr><td>Nitric Acid</td><td>VWR</td><td>bdh 3044</td><td></td></tr><tr><td>NS (Neuronal Supplement) 21&amp;nbsp;</td><td>prepared in the lab</td><td></td><td>Source: reference 69</td></tr><tr><td>Pasteur pipets, 5 &amp;frac34;&amp;rdquo;&amp;nbsp;</td><td>Fisher</td><td>13-678-6A</td><td>Use this cotton-plugged 5 &amp;frac34;&amp;rdquo; Pasteur pipette for cellular trituration. Fire-polish the tip beforehand to smooth the cut surface and to reduce the internal diameter to 50-80% of the original. Too small a tip will disrupt the cells and reduce cell viability, but too large a tip will decrease the efficiency of trituration.</td></tr><tr><td>Pasteur pipets, 9&amp;rdquo;&amp;nbsp;</td><td>Fisher</td><td>13-678-6B</td><td></td></tr><tr><td>Potassium chloride (KCl), SigmaUltra, &amp;ge;99.0%</td><td>Sigma-Aldrich</td><td>P9333-500G</td><td></td></tr><tr><td>Serological pipet, 2 ml</td><td>BD falcon</td><td>357507</td><td></td></tr><tr><td>Serological pipet, 5 ml&amp;nbsp;</td><td>BD falcon</td><td>357543</td><td></td></tr><tr><td>Serological pipet, 10 ml</td><td>BD falcon</td><td>357551</td><td></td></tr><tr><td>Serological pipet, 25 ml</td><td>BD falcon</td><td>357525</td><td></td></tr><tr><td>Serological pipet, 50 ml&amp;nbsp;</td><td>BD falcon</td><td>357550</td><td></td></tr><tr><td>Sodium bicarbonate (NaHCO3, Sodium hydrogen carbonate), SigmaUltra, &amp;ge;99.5%</td><td>Sigma-Aldrich</td><td>S6297-250G</td><td></td></tr><tr><td>Sodium chloride (NaCl), SigmaUltra, &amp;ge;99.5%</td><td>Sigma-Aldrich</td><td>S7653-250G</td><td></td></tr><tr><td>Sodium hydroxide (NaOH), pellets, 99.998% trace metals basis</td><td>Sigma-Aldrich</td><td>480878-250G</td><td></td></tr><tr><td>Sodium phosphate dibasic heptahydrate (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;(7H&lt;sub&gt;2&lt;/sub&gt;O)), &amp;ge;99.99%, Aldrich</td><td>Sigma-Aldrich</td><td>431478-250G</td><td></td></tr><tr><td>Sucrose, SigmaUltra, &amp;ge;99.5% (GC)</td><td>Sigma-Aldrich</td><td>S7903-250G</td><td></td></tr><tr><td>Syringe filter, sterile, 0.2 &amp;micro;m</td><td>Corning</td><td>431219</td><td></td></tr><tr><td>Syringe, 3 ml</td><td>BD falcon</td><td>309585</td><td></td></tr><tr><td>Transferrin, Holo, bovine plasma</td><td>Calbiochem</td><td>616420</td><td></td></tr><tr><td>Trypan Blue stain, 0.4%</td><td>GIBCO-Invitrogen</td><td>T10282</td><td>This is used for counting live/dead cells. Renew an old trypan blue solution if it is re-used many times (&lt;em&gt;e.g.&lt;/em&gt; several times a week for several weeks), because it will form precipitates and result in erroneous readouts of cellular density.</td></tr><tr><td>Trypsin, type XI</td><td>Sigma-Aldrich</td><td>T1005-5G</td><td></td></tr><tr><td>Trypsin-EDTA solution, 0.25%&amp;nbsp;</td><td>GIBCO-Invitrogen</td><td>25200-056</td><td></td></tr><tr><td>Uridine</td><td>Sigma-Aldrich</td><td>U3003-5G</td><td>Stock solution is prepared at 50-mg 5-fluoro-2&#039;-deoxyuridine and 125 mg uridine in 25 ml DMEM (8.12 and 20.48 mM, respectively).</td></tr><tr><td>REAGENTS - immunocytochemistry</td><td></td><td></td><td></td></tr><tr><td>Antibody, rabbit polyclonal anti-MAP2</td><td>Merck Millipore</td><td>AB5622</td><td></td></tr><tr><td>Antibody, mouse monoclonal anti-GFAP cocktail</td><td>Merck Millipore</td><td>NE1015</td><td></td></tr><tr><td>EQUIPMENT - tattooing</td><td></td><td></td><td></td></tr><tr><td>AIMS</td><td>Animal Identification and Marking Systems, Inc.</td><td>NEO&amp;#8211;9</td><td>This Neonate Rodent Tattooing System is an electric system that works by rapidly moving 1- or 3-point tattoo needles vertically into the skin. Activate the tattoo machine once for approximately 0.5 sec, while the tattoo needle tips are kept perpendicular to the skin surface. We prefer three-needle tattooing to maximize the tattooed area, but one-needle tattooing is effective on narrower areas, &lt;em&gt;e.g.&lt;/em&gt; the toes, or if fine mechanical control is necessary, &lt;em&gt;e.g.&lt;/em&gt; when numbers are tattooed. Two rounds of tattooing at the slowest speed (setting &quot;1&quot; out of 3 steps) are typically sufficient to produce a visible and long-lasting tattoo of the paw pads.</td></tr><tr><td>EQUIPMENT - genotyping</td><td></td><td></td><td></td></tr><tr><td>Electrophoresis system, horizontal, Wide Mini&amp;#8211;Sub Cell GT</td><td>BIO&amp;#8211;RAD</td><td>170&amp;#8211;4405</td><td>Typical electrophoresis parameters are electrical field strength at 6 V/cm and 25 min duration for a 10 cm gel.</td></tr><tr><td>FluorChem 8800</td><td>ProteinSimple</td><td>FluorChem 8800</td><td></td></tr><tr><td>PCR, MJ Mini Thermal Cycler</td><td>BIO-RAD</td><td>PTC-1148EDU</td><td>Our PCR reactions for the Tor1a gene in &amp;#916;E-torsinA knock-in mice are as follows: 1 cycle of denaturation at 94 &amp;#176;C for 3 min, 35 cycles of denaturation at 94 &amp;#176;C for 30 sec, annealing at 58 &amp;#176;C for 30 sec, extension at 72 &amp;#176;C for 2 min. This is followed by final extension at 72 &amp;#176;C for 10 min, and holding at 4 &amp;#176;C.</td></tr><tr><td>Power supply, PowerPac Basic</td><td>BIO-RAD</td><td>164-5050</td><td></td></tr><tr><td>EQUIPMENT - cell culture</td><td></td><td></td><td></td></tr><tr><td>Automated cell counter, Countess</td><td>GIBCO-Invitrogen</td><td>C10310</td><td>This automated cell counter separately measures the densities of live and dead cells (non stained and stained by trypan blue, respectively). It is important to know the optimal range of density measurements: the counter that we use has the highest accuracy in the range from 1 x 10&lt;sup&gt;5&lt;/sup&gt; to 4 x 10&lt;sup&gt;6&lt;/sup&gt; cells/ml. If the measured cell density values fall outside the recommended range, adjust the resuspension volume appropriately.</td></tr><tr><td>Biological Safety Cabinet, Class II, Type A2</td><td>NUAIRE</td><td>NU-425-400</td><td>This hood is used for all cell culture procedures, except for brain dissection.</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Incubator, AutoFlow, Humidity Control Water Jacket</td><td>NUAIRE</td><td>NU-4850</td><td></td></tr><tr><td>Horizontal Clean Bench</td><td>NUAIRE</td><td>NU-201-330</td><td>This clean bench is used for brain dissection (steps 3.1.1 and 3.1.2 of &quot;Brain Dissection and Cellular Dissociation)&quot;.</td></tr><tr><td>Orbit LS Low Speed Shaker</td><td>Labnet</td><td>S2030-LS-B</td><td></td></tr><tr><td>SORVALL RC-6 Plus Superspeed Centrifuge</td><td>Fisher</td><td>46910 (centrifuge)/46922 (rotor)</td><td></td></tr></tbody></table>

rapid genotyping of animals followed by establishing primary cultures of brain neurons

哺乳动物神经元的形态和功能的高分辨率分析往往需要个体动物随后的神经元的原代培养的分析的基因分型。我们描述了一套程序:标记新生小鼠进行基因分型，快速基因分型，并建立从这些小鼠脑的神经元的低密度培养物。个别小鼠通过纹身，其允许长期持久的识别到成年标记。基因分型通过所述协议是快速和有效的，并且允许进行核酸具有良好的可靠性的自动提取。这是根据情况下有足够的时间对常规的基因分型是不可用， 例如 ，是有用的，在于从新生致死遭受小鼠。在低浓度，这使得成像实验以高的空间分辨率产生原代神经元培养物。这种培养方法需要胶质滋养层的准备之前，神经元电镀。在protocol是其全部施加到运动障碍DYT1肌张力障碍（ΔE-torsinA敲除小鼠）的小鼠模型中，与神经元培养物从这些小鼠的海马，大脑皮质和纹状体制备。该协议可以被应用到小鼠与其他的基因突变，以及对其他物种的动物。此外，可用于分离子项目的协议的各个组件。因此，该协议将有广泛的应用，不仅在神经科学，而且在生物科学和医学等领域。

作为该协议的应用的一个例子，有代表性的结果示于通过纹身，可靠的基因分型的各种实验条件下标记的小鼠，并且对神经胶质饲养层建立初级神经元培养物。 纹身 新生幼仔被标记上（ 图1中"新生儿"），使用纹身系统的爪垫。标签保持3周清晰可见（&#39;3周龄&#39;）和32周龄（&#39;32周龄&#39;）。个别小鼠可通过在四只爪子（编号1...

Watch this Scientific Journal Video about Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons at JoVE.com

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

我们描述了标签和基因分型新生小鼠，并从中产生的主要神经文化的过程。基因分型是快速，有效和可靠的，并且允许进行自动核酸提取。这是为neonatally致死小鼠和其文化，需要事先完成基因分型的特别有用的。

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 ...

rapid-genotyping-animals-followed-establishing-primary-cultures-brain

Research

JoVE Journal

Neuroscience

16.2K 次观看.  University of Iowa Carver College of Medicine. 我们描述了标签和基因分型新生小鼠，并从中产生的主要神经文化的过程。基因分型是快速，有效和可靠的，并且允许进行自动核酸提取。这是为neonatally致死小鼠和其文化，需要事先完成基因分型的特别有用的。 

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

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

评定神经连接的形成在共培养体系的光遗传学方法

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

科研

发育生物学

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 ...

神经科学

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

诱导多能干细胞的快速神经元分化有关微电极阵列测量网络活动

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.
Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA+ neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor1. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

神经元的初步培养体系的研究疱疹病毒潜伏期和活化

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

免疫与感染

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) would provide a powerful platform for neuroscientists to investigate axo-glial interactions, properties and differentiation of multipotent progenitors, and progression of oligodendroglial lineage cells at the cellular and molecular level.  We describe here a CNS aggregate culture system from embryonic rat forebrains, which can be maintained in a serum-free medium up to 3-4 weeks and is used in our laboratory as a model to study neuron-glia interaction and CNS myelination.  This video clip will demonstrate how to isolate and grow these CNS aggregate cultures from E16 rat brain.  Furthermore, from the same brain dissection, highly enriched regular dissociated neuronal cultures can be readily obtained and used for various studies on CNS neurons or used for co-cultures with other cells. 

A protocols for an embryonic rat brain aggregate culture system is described. Multipotent progenitors in the aggregates can develop and differentiate into neurons, astrocytes and oligodendrocytes.

制备大鼠脑组织神经元和神经胶质发展研究总结文化

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) ...

生物学

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 ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

从E14-E16斯普拉格·道利大鼠胚胎分离的混合原发希马和皮质神经元生成神经球

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

胚胎小鼠脑混合细胞培养物研究感染和先天免疫

Establishing Mixed Neuronal and Glial Cell Cultures from Embryonic Mouse Brains to Study Infection and Innate Immunity

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of neurons, astrocytes, oligodendrocytes, and microglia. Due to increasing efforts to replace and reduce animal use, a variety of in vitro cell culture systems have been developed to explore innate cell properties, which allow the development of therapeutics for CNS infections and pathologies. Whilst certain research questions can be addressed by human-based cell culture systems, such as (induced) pluripotent stem cells, working with human cells has its own limitations with regard to availability, costs, and ethics. Here, we describe a unique protocol for isolating and culturing cells from embryonic mouse brains. The resulting mixed neural cell cultures mimic several cell populations and interactions found in the brain in vivo. Compared to current equivalent methods, this protocol more closely mimics the characteristics of the brain and also garners more cells, thus allowing for more experimental conditions to be investigated from one pregnant mouse. Further, the protocol is relatively easy and highly reproducible. These cultures have been optimized for use at various scales, including 96-well based high throughput screens, 24-well microscopy analysis, and 6-well cultures for flow cytometry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. This culture method is a powerful tool to investigate infection and immunity within the context of some of the complexity of the CNS with the convenience of in vitro methods.

作者聚焦:从胚胎小鼠大脑中建立混合神经元和神经胶质细胞培养物以研究感染和先天免疫  

This protocol presents a unique way of generating central nervous system cell cultures from embryonic day 17 mouse brains for neuro(immuno)logy research. This model can be analyzed using various experimental techniques, including RT-qPCR, microscopy, ELISA, and flow cytometry.

从胚胎小鼠大脑中建立混合神经元和神经胶质细胞培养物以研究感染和先天免疫

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of ...

动物的基因分型快速其次是建立大脑神经元原代培养

摘要

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此视频中的章节

评定神经连接的形成在共培养体系的光遗传学方法

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

诱导多能干细胞的快速神经元分化有关微电极阵列测量网络活动

神经元的初步培养体系的研究疱疹病毒潜伏期和活化

制备大鼠脑组织神经元和神经胶质发展研究总结文化

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

从E14-E16斯普拉格·道利大鼠胚胎分离的混合原发希马和皮质神经元生成神经球

从胚胎小鼠大脑中建立混合神经元和神经胶质细胞培养物以研究感染和先天免疫

从胚胎小鼠大脑中建立混合神经元和神经胶质细胞培养物以研究感染和先天免疫

从胚胎小鼠大脑中建立混合神经元和神经胶质细胞培养物以研究感染和先天免疫