在涉及的海兔神经细胞培养实验室工作的经费由美国国立卫生研究院的清单R01和美国国立卫生研究院R21MH077921。

准备（参见解决方案在协议结束部分解决方案的组成） <ol><li>准备培养皿。涂层玻璃Mattek玻璃底培养皿与聚- L -赖氨酸（四硼酸钠）。加入足够的完全覆盖的玻璃和离开&gt; 1小时（可以过夜）。彻底清除聚- L -赖氨酸，在人工海水冲洗（ASW）的4-5倍。删除最后一次冲洗后，加2毫升50％L15（补充盐和含L -谷氨酰胺在终浓度为2mm）/ 50％血淋巴。这种培养基中必​​须至少一个小时，在盘前电镀细胞，使血淋巴大衣的菜。 </li><li>清洁工具和Sylgard菜用乙醇，冲洗彻底DDH 2 0&gt; 1小时，然后将他们文化罩在UV光下。 </li><li>准备的神经元显微切割电极是雪亮的。 使用微电极拉马（如萨特火焰山布朗的P - 97），外直径1.5毫米，内径0.86至1.12毫米和100毫米的长度与拉玻璃吸管电极（如AM系统目录＃628000;世界精密仪器TW150 - 4，1.55 / 1.12）。使用参数产生长如此之高的阻力和束状的精尖，没有毛细管流体摄入量时，在溶液中的电极。流体半月板的存在，意味着在隔离，电极尖端会损害神经元。萨特电极食谱设立电极方案提供了出色的指引。我们用一个框丝，和一个单步拉来生成文化的电极。 </li><li>准备麻醉的解决方案（0.35M氯化镁），培养基（L15辅以盐和血淋巴），和蛋白酶溶液（1％蛋白酶类型第九，Sigma公司，1个单位/毫克，到终浓度为10单位/ ml）。 </li><li>动物：成人80-100克海兔感觉神经元的隔离和LFS的运动神经元。使用隔离L7运动神经元的少年1-4 克海兔。轻轻取出动物从海水中的坦克，并维持在海水中不超过30分钟（在烧杯中，桶或塑料袋），然后才开始麻醉和文化的过程。 </li></ol>文化程序 A.培养胸腔80-100克海兔感觉神经元<ol><li>清除神经节麻醉的动物：成年海兔 （80-100克）18号1.5寸毫针用60毫升注射器注入0.35米氯化镁2。输入动物的脚在约35度的角度，并没有进入过深。我们的目标是不穿透五脏六腑注入到动物hemocoel。动物应该成为非常扩张和宽松的。 </li><li>解剖神经节：引脚与麻醉动物解剖盘，在头部和尾部的针（18号针头的工作以及为80-100克动物），脚朝上。用有齿镊保持脚和穿过皮肤和底层的结缔组织脚长度（从头部到尾部）用手术剪刀。引脚的双方。使用镊子和剪刀，切断食管，拉边给了暴露的神经节。用细镊子和罚款剪刀，剪出的胸腔踏板神经节（在胸膜神经节感觉神经元）。离开一个公平的神经，因为这使得更容易牵制蛋白酶处理后的神经节。 </li><li>蛋白酶消化，神经节：将神经节蛋白酶溶液（10 mg / ml的1个单位/毫克L15）在孵化器设定在34.5 ° C（34-35 ° C是可以）2小时和15分钟。使用精尖镊子转移和洗反潜神经节的三倍。保持在L15的神经节。需要注意的是蛋白酶孵育时间可以有所不同。的目的是为了让结缔组织可以很容易地删除。如果实在是太难了desheath不损坏的神经元的神经节，增加蛋白酶孵育时间。如果它是非常容易desheath的神经节，神经元是“软”，减少蛋白酶孵育时间。 </li><li> Desheathing神经节：蛋白酶消化，神经节转移到60毫米或35mm菜含有sylgard和L15。下一个立体显微镜（外部卤素灯照明）查看，删除踏板节和牵制在正确的方向用昆虫针和罚款钳Sylgard菜胸膜神经节。用细镊子和一个万​​纳手术剪刀，小心地抬起结缔组织和削减它暴露的感觉集群。要小心不要过接触或损坏的神经元somata。从盘删除任何多余的结缔组织，并添加更多的介质，确保从未desheathed节暴露在空气中。感觉集群由约200聚集直径为40-50微米的神经元。设置PIN，使在很短的距离从gangl的“后”离子（视野范围内的）。 </li><li>神经元的分离：使用长，锋利的玻璃文化电极，拔出所确定的神经元的一个个感人只是偏离中心的细胞体（不刺穿），缓慢而稳定地拉动细胞SOMA，连同轴突，远离神经节。轻轻拍打对引脚逐出电极的神经元。 </li><li>电镀神经元：使用pipetman（P10）Sylgard菜转移到培养皿的单个神经元。在转移到塑料尖，使涂层与血淋巴提示神经元，吸管培养基（这可防止粘塑料的神经元）。非常谨慎，以避免暴露任何气泡或任何极端势力（即轻轻拿起，并从pipetteman移除神经元）的神经元。整个菜均匀分布的神经元。用锋利的电极轻轻伸直的过程，挖掘他们下到谷底。 </li><li>孵化：留在室温下不超过3个小时的菜在显微镜阶段。我们经常让他们在一夜之间。确保显微镜阶段，在此期间不受干扰。盖用铝箔，以防止光的阶段。轻轻将它们传输到18 ° C培养箱。 </li><li>文化应坚持的菜约3小时内，新突起的生长，应约6-12小时内可见。孤立的感觉神经元的增长达到3或4格高原。请注意，孤立的感觉神经元能源部没有形成autapses或与另一个化学突触，虽然他们形成电气间隙路口。 </li></ol> B.感觉神经元运动神经元共同培养的制备文化目标的运动神经元的感觉神经元形成突触。最常用的运动神经元LFS的运动神经元和L7的运动神经元从腹神经节。 LFS的运动神经元是孤立的成人（80-100克）， 海兔 ，有大约20 LFS的运动神经元，每腹神经节。 L7的运动神经元是从少年（1-4克）动物隔离，并有腹腔神经节的每一个L7的。 LFS的运动神经元，直径40-50微米，靠近腹腔神经节的腹面虹吸神经根感觉神经元的LE之间穿插，特点是每个SOMA一个微妙的暗色素斑。 L7的运动神经元是100-150微米，直径是背表面上的神经节，神经节左侧中间的边缘，目前。虽然这是L7的运动神经元的大尺寸更经济地使用LFS的运动神经元，是有利的一些实验。 L11的运动神经元也对背腹神经节，尾椎到L7的左侧面，是一个非靶运动神经元，并可以有效地控制感觉神经元fasciculates，但没有形成化学突触。 <ol><li>麻醉动物，去除神经节。对于LFS的运动神经元，胸腔感觉神经元。 L7运动神经元，0.35米MgCl 2的少年海兔 （1-4克）用21号针头使用10毫升注射器注入。 </li><li>解剖腹腔神经节：如上所述（21表针使用动物幼仔）引脚麻醉动物解剖盘。用细镊子和罚款剪刀，剪出腹神经节。 </li><li>蛋白酶消化，神经节：这是做胸腔感觉神经元的描述，除了消化时间缩短为1小时45分钟，从少年（1-4克）动物神经节。 </li><li> Desheathing神经节：35或60毫米菜含有sylgard和L15的蛋白酶消化，神经节转移。查看下一个立体显微镜（外部卤素灯照明），在神经节Sylgard菜用昆虫针和罚款钳在适当的方向。用细镊子和一个万​​纳手术剪刀，小心地抬起结缔组织和削减它暴露的神经细胞团体。为了隔离LFS运动神经元，针节，使腹面朝上，并用钳拉结缔组织鞘包含LFS desheath的运动神经元（在你的右边），揭露整个神经节半神经节的其余部分，从菜中删除任何结缔组织，并添加更多的L15。要隔离的7层或L11运动神经元，针的神经节，使背水面朝上，并用钳拉结缔组织鞘揭露L7和L11（你的左边）节的一半。要小心不要过接触或损坏的神经元somata。将一根针，使“邮报”在从神经节的短距离（视野内）。 </li><li>神经细胞的分离：删除描述了一个尖锐的电极为感觉神经元的神经元。 LFS的神经元是差异erentiated从邻近的LE他们somata一个小暗色素斑的基础上的感觉神经元。 L7的神经元可以有所区别，因为L11是L11的神经元首先从尾椎到L7，因为L11的细胞体是椭圆形的形状，而L7细胞体是通才，因为L11的轴突往往分为两个收盘跳转到该单元格身体。 </li><li>电镀神经元：使用pipetman（P10）Sylgard菜转移到培养皿中的单个神经元，感觉神经元中所述。 </li><li>感觉神经元的配对：让我们坚持至少1小时的运动神经元的培养皿中。然后添加一个pipetman孤立的感觉神经元，提供每一个镀金的运动神经元附近的感觉神经元。使用锋利的玻璃电极认真理顺感觉神经元轴突，哄的感觉细胞体内的运动神经元的位置，在距离等于或略高于感觉细胞轴突的长度，。使用玻璃电极，同轴感觉轴突接触到的运动神经元，轻轻使用的锥形电极一侧终于有了感觉轴突身体接触的运动神经元轴突。不要解除培养皿中，而是轻轻滑动盘沿显微镜阶段。 </li><li>留在室温不低于3小时，为感觉神经元转移到18 ° C培养箱显微镜舞台上的菜肴。 </li><li>文化应坚持的菜约3小时内，新突起的生长，应约6-12小时内可见。感觉神经元的形成与运动神经元谷氨酸突触非常迅速 - 只要细胞是贴壁，足以执行尖锐的电极记录（3-5小时），可以记录兴奋性突触后电位。突触连接继续增加，直到3格，然后直到DIV7稳定。神经元，应显示在第一个3天的文化精心制作的突起生长。 </li></ol> C.血淋巴的制备淋巴是用来作为一个在海兔文化的生长因子（类似于使用哺乳动物细胞培养胎牛血清） 。它收集了从大（500克，1公斤）的动物，收集血淋巴（闲谈）的最佳时间是在春季（3月中旬至6月）。襁褓使只有一小部分动物暴露在一个一次性underpad的动物，清洁，皮肤用乙醇的部分，然后拿着它，而另一个人使用无菌刀片切口，暴露面积。到一个干净的烧杯中，使血淋巴鞘，确保它不接触动物的脏皮肤，挤出的动物。血淋巴中填充的动物hemocoel（即动物基本上是一个血淋巴囊）。重新包装的动物一次或两次，使一个新的切口，挤压，以收集尽可能多的血淋巴（收集在一个新的烧杯中，因此，如果它被污染，首先收集仍然是可用的）。从每个动物的血淋巴应分开存放（即不从不同的动物池血淋巴）。在2000年为10分钟XG旋转血淋巴去除血细胞。上清分装在10毫升分装，标签由动物和存储于-80 ° C。请注意，储存在-20 ° C的血淋巴的形式在介质中沉淀。每次培养基准备，必须使用的血淋巴中的一个新等分，等分应解冻前的准备中期。不要解冻后重新冷冻。

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海兔感觉运动文化的成功做好准备，有一个有点慢的学习曲线，因为它涉及到显微切割和操纵通过一个立体显微镜观察单个神经元的精细动作技能的发展。根据我们的经验，它需要大约1-3周的实践，获得健康的文化孤立的感觉神经元和一个额外的1-3周，以了解如何对感觉神经与运动神经元。我们经常准备在Labconco超净工作台文化，虽然有可能准备任何实验室的工作台或办公桌上，只要显微?...

为文化需要的解决方案<ol><li> 0.35米氯化镁2，在室温下储存，使用麻醉。 </li><li>聚- L -赖氨酸的解决方案，西格玛P - 1524兆瓦&gt; 300,000，0.1M硼酸钠pH8.2至0.5mg/ml的解决方案。涡和过滤通过0.22微米的过滤消毒，存放在4C °。不要冻融。 </li><li> L - 15的中等粉（莱博维茨）（Sigma公司：L4386）与盐的补充，使1公升 <table border="1" cellspacing="0" cellpadding="0"><tr><td> L - 15粉</td><td> 13.8克</td></tr><tr><td>氯化钠</td><td> 15.4摹</td></tr><tr><td> D -葡萄糖</td><td> 6.24摹</td></tr><tr><td>硫酸镁4 7H 2 0 </td><td> 6.45克</td></tr><tr><td>氯化钾</td><td> 350毫克</td></tr><tr><td>碳酸氢钠3 </td><td> 170毫克</td></tr><tr><td>氯化镁2 · 6H 2 O </td><td> 5.49摹</td></tr><tr><td>氯化钙2 · 2H 2 O </td><td> 1.43克</td></tr><tr><td> HEPES </td><td> 3.53克</td></tr></table>添加ddH20到1升。 pH值7.4-7.5左右，应添加100X笔/链球菌解决方案10毫升，通过一个0.22米过滤器和过滤器消毒。在4C0商店不超过1个月。 </li><li>蛋白酶消化解决方案：1％蛋白酶IX（1unit/mg）L15（辅以如上盐）或反潜立即使用前，通过一个0.22米的微孔过滤灭菌。 5mls应该足够了两种动物的神经节（确保神经节是完全沉浸在蛋白酶溶液）。适马报告说，他们将停止销售蛋白酶第九。替代蛋白酶是：Dispase II（罗氏应用科学部目录＃04942078001）。 </li><li>培养基。前准备文化，解冻10毫升的血淋巴等份和混合10 L15的MLS（辅以如上盐），使20mls培养基。加入200μL200毫米L -谷氨酰胺，拌匀，用于准备文化。这种介质应准备新鲜每次文化。 </li><li>人工海水可从即时海洋（水族馆系统，导师，OH）如下：450毫米氯化钠，氯化钾10毫米，30毫米2（H 2 O）氯化镁， 硫酸镁 20毫米，10毫米氯化钙2（2H 2 O），10毫米的HEPES，pH值，调整至7.4。 </li></ol>

preparation of aplysia sensory-motor neuronal cell cultures

海洋软体动物海兔californica的神经系统是相对简单的，约20,000神经元组成。神经元大（直径可达1毫米）和识别，具有不同的大小，形状，位置和色素沉着，胞体外部暴露在五个配对神经节分布于整个动物的身体。这些属性允许划定的基本电路 1，在动物的特定行为的调查。感觉和运动神经元之间的突触连接，是一个在动物的鳃撤退反射的核心组成部分，其中动物撤回其刺中虹吸触觉刺激的一个简单的防御反射。这种反射的非关联和关联学习经历的形式，包括敏，习惯性和经典性条件反射，。感官电机突触突触可塑性的研究特别的好处，可以在文化重组，特点刺激引起的可塑性在动物的 2,3行为有直接关联的形式。具体来说，羟色胺的应用程序产生一个突触的加强，取决于应用协议，持续几分钟（短期便利），小时（中期的便利）或天（长期便利）。相比之下，肽发射机FMRFamide的应用产生了突触减弱或抑郁症，取决于应用协议，去年从天分钟（长期抑郁症）。的神经元的大尺寸允许反复突触强度的急剧电极记录天期间注射表达载体，siRNA和其他化合物，针对特定的信号级联和分子，从而确定分子和细胞生物学的步骤背后的变化在突触效能。 海兔文化体系的一个额外的好处，从事实的神经元突触特异性展示文化4,5。因此，感觉神经元不与自己（autapses）或与其他感觉神经元形成突触，也没有形成与非目标确定在文化运动神经元突触。静脉曲张，感觉和运动神经元之间的突触联系的网站，都足够大（直径在2-7微米），允许在光镜水平研究目标的运动神经元突触的形成与（以及突触形态的变化）。 在这段视频中，我们展示了准备的感觉，运动神经元的文化，包括anesthetizing成人和青少年海兔的每一步，剖析他们的神经节，神经节蛋白酶消化，去除结缔组织显微切割，感觉和运动神经元的识别和清除每个显微切割细胞类型，运动神经元，感觉神经元和感觉神经轴突的操纵此外电镀形成与培养运动神经元的联系。

Watch this Scientific Journal Video about Preparation of Aplysia Sensory-motor Neuronal Cell Cultures at JoVE.com

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

小学文化<em&gt;海兔</em&gt;感觉运动神经元突触的形成和突触可塑性研究提供一个模型的制备<em&gt;在体外</em&gt;。这个视频演示的感觉和运动神经元的鉴定和显微切割<em&gt;海兔</em&gt;神经节以及建立和保持文化的感觉运动神经元的方法。

制备海兔感觉运动神经元细胞培养

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

preparation-of-aplysia-sensory-motor-neuronal-cell-cultures

Research

JoVE Journal

Biology

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

15.9K 次观看.  University of California, Los Angeles. 小学文化海兔感觉运动神经元突触的形成和突触可塑性研究提供一个模型的制备在体外。这个视频演示的感觉和运动神经元的鉴定和显微切割海兔神经节以及建立和保持文化的感觉运动神经元的方法。

视频: Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

ES Cell-derived Neuroepithelial Cell Cultures

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In general, the differentiation of ES cells follows the concept to first generate immature progenitor cells, which then can be propagated and differentiated into mature cellular phenotypes. This also applies for ES cell-derived neurogenesis, in which the development of neural cells follows two major steps: First, the derivation and expansion of immature neuroepithelial precursors and second, their differentiation into mature neural cells. A common method to produce neural progenitors from ES cells is based on embryoid body (EB) formation, which reveals the differentiation of cells from all germ layers including neuroectoderm. An alternative and more efficient method to induce neuroepithelial cell development uses stromal cell-derived inducing activity (SDIA), which can be achieved by co-culturing ES cells with skull bone marrow-derived stromal cells (1). Both, EB formation and SDIA, reveal the development of rosette-like structures, which are thought to resemble neural tube- and/or neural crest-like progenitors. The neural precursors can be isolated, expanded and further differentiated into specific neurons and glia cells using defined culture conditions. Here, we describe the generation and isolation of such rosettes in co-culture experiments with the stromal cell line MS5 (2-5).

Derivation of neuroepithelial precursors from embryonic stem (ES) cells using stromal cell-derived inducing activity (SDIA).

ES细胞源性神经上皮细胞培养

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In ...

科研

生物学

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

从晚期果蝇蛹的大脑原发性神经文化

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

从Midgastrula舞台果蝇胚胎神经文化的制备

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

游离的小鼠皮层神经元文化的制备

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

从海兔的神经细胞培养的高分辨率成像生长锥

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

小鼠表皮神经嵴干细胞（EPI的非公务员合约雇员）文化

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

2 dGuo治疗胸腺器官培养的制备

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for neuronal survival and function. Neurotrophins are specifically required to maintain the survival and differentiation of innervating sensory neurons but the question of how these target-derived neurotrophins communicate to the cell body of innervating neurons has been an area of active research for over 30 years. The most commonly accepted model of how neurotrophin signals reach the cell body proposes that signaling endosomes carry this signal retrogradely along the axon. In order to study retrograde transport, a culture system was originally devised by Robert Campenot, in which cell bodies are isolated from their axons. The technique of preparing these compartmented chambers for culturing sensory neurons recapitulates the selective stimulation of neuron terminals that occurs in vivo following release of target-derived neurotrophins. Retrograde signaling events that require long-range microtubule dependent retrograde transport have important implications for the treatment of neurodegenerative disorders.

Here we describe the technique of preparing and maintaining compartmented chambers for culturing sensory neurons of the dorsal root ganglia.

隔间文化背根神经节神经元的制备及维修

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

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) ...