培养前一天的准备工作： <ul><li>准备无菌解剖解决方案（DS）。 </li><li>准备NBM/B27（Neurobasal Mediom B27的补充）。 </li><li>高压灭菌器DDH 2 O和消毒玻璃coversilps，如果需要的话。 </li><li>涂层组织培养皿或D -聚赖氨酸的盖玻片。 </li></ul> D -聚赖氨酸涂层： 准备无菌条件下培养前一天。 <ul><li>解冻分装10X PDL和冰的地方。 </li><li>添加1毫升的客运专线和9 ml无菌超过滤后的水拌匀（1X）。 </li><li>涂层表面与1X的PDL在室温如下过夜： <ul><li>盖玻片（Bellco玻璃，猫＃1943-00012）：75μL/盖玻片 或</li><li> 24孔培养板：300μL/孔 或</li><li> 35mm培养皿中的：1ml/dish </li></ul></li><li> （使用无菌巴斯德吸液管吸液）用无菌水冲洗5倍。 </li><li>除去水，直到表面完全干燥。 </li></ul>培养过程（在层流罩无菌条件下进行） <ol><li>到切片Vibraslicer（坎普登仪器有限公司）的支持，使用强力胶块切出的琼脂块和胶水。 </li><li>当使用萌芽阶段后期（E17 - 18）的小鼠胎儿，人道毁灭大坝，从胚胎麻袋中移除子宫和个人自由的胎儿。将胎儿放入无菌培养皿，继续概述如下。 </li><li>杀头小鼠胎儿或小狗（遵循的准则，您的机构的动物护理和使用委员会批准）。 </li><li>一个充斥着寒冷的DS60毫米培养皿菜的Whatman滤纸盘上取下皮肤和颅骨和地方脑。 </li><li>使用无菌刀片切断小脑。 </li><li>拿起大脑使用抹刀和沥干多余的液体在滤纸上，然后转移大脑Vibraslicer支持块和胶脑（尾鳍朝上，腹侧部分面临琼脂）。 </li><li>填充用冷水DS室。设置速度选择8-9。 </li><li>切200-400微米的部分，从嗅球开始。一旦刀片的Vibraslicer进入皮层，开始切割600μm冠状切片。 E17 - 18的小鼠大脑中通常会产生3-4可用切片，而P0小鼠大脑产生4-5可用切片。 </li><li>大脑切片转移到35毫米培养皿标记的DS 2使用反向端插入10毫升玻璃巴斯德吸管。 </li><li>解剖皮质和消除使用玻璃毛细管拉针的脑膜。 </li><li>切成小块，长度在0.5-1毫米的皮层外皮。 </li><li>过滤器通过0.2微米ES的过滤器附加到35毫米的培养皿中，以5毫升注射器。 </li><li>皮层组织块转移到培养皿中筛选胚胎干。 </li><li>在37 ° C下30分钟。 </li></ol>在此期间： <ol><li>清理油烟机，Vibraslicer和解剖工具。 </li><li>一个60毫米的培养皿中加入5毫升的温暖NBM/B27。 </li></ol> 30分钟后。潜伏期： <ol><li>组织转移至10毫升的DS。 1分钟让定居。 </li><li>组织转移到第一喜管的漩涡，让定居2-3分钟。 </li><li>转移到第二高。重复上面。 </li><li>转移到第一李。重复上面。 </li><li>转移到第二个李。重复上面。 </li><li>转移到第三栗。重复上面。 </li><li>转移组织与媒体的60毫米菜。 </li></ol>解剖显微镜下： <ol><li>从组织的清洁碎片，每片磨碎，通过拉玻璃吸管（使用降低孔径）轻轻掠过放松组织。 </li><li>将细胞悬液15毫升锥形管中NBM + B27的其余部分（共13毫升24孔板或11毫升5 - 35毫米菜，轻轻混匀大块组织，等待解决）。 </li><li>转移细胞悬液（0.5毫升/ 24孔板或2毫升/ 35毫米培养皿）。 </li><li>当使用盖玻片，加入80μl的细胞悬液，每盖玻片，并允许在组织培养箱内培养1小时，细胞坚持之前充斥着更多NBM/B27媒体室。 </li><li>保持在37 ° C和5％的CO 2的文化。 </li></ol>第二天 24孔板：饲料每口井用0.5毫升空调NBM/B27介质的非神经元细胞（cNBM/B27准备cNBM/B27中等议定书“下方出现） 35毫米菜：cNBM/B27介质更换0.5毫升。 保持新鲜cNBM/B27每2-3天更换中等0.5毫升文化。 虽然文化传媒不促进神经胶质细胞的增殖，文化可以被视为在文化3-5天，以进一步降低神经胶质细胞的数量，如果需要的话5μMFDU（5 - 氟-2&#39; - 脱氧尿苷，西格玛F0503）。 设置<html>为夹层和文化解剖工具，在70％乙醇消毒或高压灭菌： <ul><li>剪（中型和小型）铲（中型和小型） </li><li>镊子（中型和小型）刀片</li><li> vibraslicer缓冲区浴之刃</li><li>脑刀片支持的小金属楔</li></ul>其他材料： <ul><li>培养皿（60毫米） </li><li>培养皿（35毫米） </li><li>滤纸</li><li>玻璃吸管10毫升</li><li>一次性无菌吸管，5,10和25毫升</li><li> 0.2微米的过滤器，注射器</li><li>注射器5毫升</li><li>离心管中，15毫升</li><li>无菌巴斯德玻璃吸管</li><li>牙周膜涂层的培养皿或盖玻片</li></ul>标签六个15毫升离心管中，并按照编制： 管＃1：酶液： 50 U到5毫升的DS含有木瓜蛋白酶（沃辛顿LS 03126）： <ul><li> 100μL的L -半胱氨酸（0.8mg） </li><li> 7μL0.1N氢氧化钠</li><li> 50μLAPV（5MM） </li></ul>保留在室温下的解决方案清除。请注意，酶液，会出现&#39;多云&#39;首先，使用前需要明确。 管＃2和＃3：喜酶抑制剂： 3毫升DS + 300μLBSA /钛+ 30μLAPV（5MM）。 轻轻混匀，避免气泡，然后分成两个1.5毫升等分。 管＃4 - ＃6：低酶抑制剂： 8毫升DS + 80μLBSA /钛+ 80μLAPV（5MM）。 轻轻混匀，避免气泡，分成三个2.6毫升分装。 放置管编号为＃6＃2在冰上。你管＃1（酶液）在室温下。 解决方案及分装<ul><li>解决方案（缓冲液）西格玛公式WT 500毫升[浓度。] </li><li>氯化钠氯化钠的S - 9625 58.4580.0克137mM </li><li>氯化钾氯化钾的P - 4504 74.564.0克5.4mM </li><li>磷酸钠磷酸氢二铵无水钠2 HPO 4的S - 0876 142.00.24克0.17mM </li><li>磷酸二氢钾无水KH 2 PO 4个P - 5379 136.090.3克0.22mM </li></ul>称取400毫升超过滤后的水，直到解散所有的配料和混合。使终体积为500毫升。放置在一个干净的瓶子和高压灭菌。储存于4 ° C和一个标签“的DS解决方案”。 <ul><li>解决方案B（HEPES）西格玛公式WT 250毫升浓。] </li><li>羟乙基羟乙基的H - 3375 283.320.97克9.9mm的</li></ul>超过滤后的水加入200毫升。混合直到溶解，使终体积为250毫升。放置在一个干净的瓶子和高压灭菌。储存于4 ° C和标签“DS解决方案B”。 <ul><li>工作液500毫升浓] </li><li>超过滤水400毫升</li><li>联合解决方案，一个25毫升</li><li>股票B液14毫升</li><li> D（+）-葡萄糖SIGMA的G - 8270 3.0克33.3mM </li><li>蔗糖Sigma公司的S - 0389 7.5克43.8 mM的</li></ul> 1N氢氧化钠调节pH至7.4。使终体积超过滤后的水500毫升。倒出到一个干净的玻璃瓶和高压灭菌。保存在4 ° C。标签“解剖解决方案”。 D -聚赖氨酸的制备： <ol><li>在1mg/ml的准备聚- D -赖氨酸的10倍原液（PDL; Sigma公司的P - 7280）在无菌的H 2 O。 </li><li>设为1.0毫升分装。该解决方案可以储存在-20 ° C至3个月。 </li></ol> 媒体 <ol><li>加入10ml的乙- 27补编（Gibco公司17504-044），以500毫升的NBM瓶（Neurobasal培养基，GIBCO 21103-049）。 </li><li> 40毫升分装和存储4 ° C。 </li></ol> APV <ol><li>加入10 ml无菌H 2 O 10毫克APV的小瓶（2 -氨基- 5 - phosphonopentanoic酸，适马A - 5282），调匀。 </li><li>准备180μL分装并储存于-20 ° C </li></ol> BSA /钛 <ol><li>溶解在10毫升的DS 1克BSA（牛白蛋白。西格玛A - 7030）和1 g胰蛋白酶抑制剂（SIGMA的T - 9253）。 </li><li> 1N氢氧化钠调节pH至7.4。 </li><li>通过0.2微米注射器过滤器过滤消毒。 </li><li>在-20 ° C到400μL分装和存储的鸿沟</li></ol> L -半胱氨酸在Eppendorf管中，溶解在200μL的DS 0.6毫克L -半胱氨酸（Sigma公司的C - 7755）。 琼脂（4％） <ol><li> 4克细菌用琼脂（DIFCO 0140-01）溶解在100毫升的无菌水。保持在4℃直至文化需要。 </li><li>微波琼脂融化，填补了35毫米的培养皿。让我们坐降温和聚合。 </li></ol>木瓜蛋白酶（沃辛顿LS 03126） e_title“&gt;非神经文化NUNC培养瓶涂层的培养瓶（4 NUNC瓶） <ol><li>加入9 mL无菌水各3管10X的PDL 1X的PDL。 </li><li>转移30毫升1X的PDL在第一瓶。 </li><li>轻微移动，直到所有的增长表面覆盖。让我们坐了1分钟。 </li><li> 1X客运专线传输到第二瓶。让我们坐了1分钟。 </li><li>重复第三和第四瓶。 </li><li>清洗所有的四瓶150毫升无菌H 2 O 2倍</li></ol>准备酶液（ES）的： <ol><li>在0.6 mL离心管重量出0.8毫克L -半胱氨酸（Sigma公司C7755），添加150毫升DS和涡流，直到晶体溶解。 </li><li>将转移5毫升的DS到15毫升的锥形管，再加入L -半胱氨酸150毫升。 </li><li>新增50个单位的木瓜蛋白酶。 （顿LS 03126） </li><li> 7毫升0.1N氢氧化钠。 </li></ol> PREPARE的MEM（GIBCO公司＃11090-081） <ol><li>取出500毫升的纪念瓶65毫升。节省10毫升准备葡萄糖。 </li><li>添加5毫升笔/链球菌（GIBCO＃15070-063）。 </li><li>添加10毫升1M葡萄糖（Sigma公司摹- 8270）（1.8克/ 10毫升的MEM） </li><li>添加50毫升小牛血清（GIBCO＃16140-071）或牛小牛血清（欧米茄BC - 04）。 </li><li>拌匀，分装并储存于4 ° C。 </li></ol> NEUROBASAL MEDIUM/B-27补充（NBM/B27） NBM，500毫升（Gibco公司＃21103-049），B - 27（50X），10毫升（GIBCO＃17504-044） <ol><li>新增的B - 27小瓶全部内容的NBM瓶和混合。 </li><li>分装和储存在4 ° C </li></ol>脑组织解剖<ol><li>从70％乙醇清扫前干解剖工具。 </li><li>在3-15毫升管，每年转移10毫升的DS。 </li><li>选择鼠标幼仔（P0 - P3），，您将需要4瓶三个大脑。 </li><li> 35毫米培养皿加冷副局长。 </li><li>杀头鼠标小狗，并剖析了脑筋。 </li><li>放置到培养皿菜含有冷的DS大脑。 </li><li>斩件，小到足以穿过一个玻璃吸管的组织。 </li></ol>酶分离现象<ol><li>删除尽可能多的DS与巴斯德吸管盘。 </li><li>通过过滤器的ES 0.2微米的过滤器和5CC针筒。 </li><li>放置在菜用纸巾ES。 </li><li>孵育组织在37 ° C培养箱（CO 2免费）为30分钟。 </li></ol>洗涤纸巾<ol><li>传输用玻璃吸管，第一局副局长管组织，确保获得尽可能少的ES。 </li><li>在1500转离心30秒。 </li><li>转移组织和重复的过程两次。 </li></ol> TRITURATION <ol><li> MEM / FBS的38毫升放入一个50毫升离心管</li><li> MEM / FBS的加入3毫升至35毫米盘和转移组织这个培养皿。 </li><li>游离于通过1000μl小指吸管尖轻轻研制过程中的组织。 </li><li>细胞悬液转移到管内含有MEM / FBS的38毫升拌匀。 </li></ol>电镀<ol><li>直立站在培养瓶，每个瓶子得到等量的细胞悬液和媒体。 </li><li>每个培养瓶放入90毫升MEM / FBS的。 </li><li>添加10 ml的细胞悬液，在每个瓶子。 </li><li>标签：非神经元，首字母缩写和日期。孵育37 ° C和5％的CO 2。 </li></ol>喂饲<ol><li>在3天或4，与温暖的MEM / FBS（倒出所有的媒体和取代MEM / FBS的100毫升）饲料细胞。文化可能需要7-10天，成为融合。 </li><li>在7-10日变更为NBM/B27媒体。 </li><li>第二天，收集媒体。用0.22μm的过滤</li><li> 40毫升的分装和饲料用MEM / FBS的文化。 </li><li>未来2〜3天，改变NBM/B27媒体</li><li>第二天，收集并重复上述步骤。 </li><li>不断收集大约媒体。 2个星期。 </li></ol>

The authors have nothing to disclose.

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>5-Fluoro-2'-deoxyuridine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F0503</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S9625</td>
<td>&nbsp;</td>
<tr>
<td>Vibraslicer</td>
<td>&nbsp;</td>
<td>Campden Instruments Ltd.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Potassim Chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4504</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Phosphate Dibasic</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S0876</td>
<td>&nbsp;</td>
<tr>
<td>Potassium Phosphate Monobasic</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P5379</td>
<td>&nbsp;</td>
<tr>
<td>Hepes</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3375</td>
<td>&nbsp;</td>
<tr>
<td>D (+)-Glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G8270</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S0389</td>
<td>&nbsp;</td>
<tr>
<td>Poly-D-Lysine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P7280</td>
<td>&nbsp;</td>
<tr>
<td>B27 Supplement</td>
<td>&nbsp;</td>
<td>Invitrogen (Gibco)</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>Neurobasal Media (NBM)</td>
<td>&nbsp;</td>
<td>Invirtogen (Gibco)</td>
<td>21103-049</td>
<td>&nbsp;</td>
<tr>
<td>2-Amino-5-phosphonopentanoic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A5282</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Albumin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A7030</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin Inhibitor</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T9253</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Hydroxide, 1N solution</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SS266-1</td>
<td>&nbsp;</td>
<tr>
<td>L-Cysteine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C7755</td>
<td>&nbsp;</td>
<tr>
<td>Bacto Agar</td>
<td>&nbsp;</td>
<td>Difco</td>
<td>0140-01</td>
<td>&nbsp;</td>
<tr>
<td>Papain</td>
<td>&nbsp;</td>
<td>Worthington </td>
<td>LS 03126</td>
<td>&nbsp;</td>
<tr>
<td>Sterile 0.2 &micro;m Syringe Filter</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>DDA02025S0</td>
<td>&nbsp;</td>
<tr>
<td>Glass Coverslips, No1, &#216;12mm</td>
<td>&nbsp;</td>
<td>Bellco Glass Inc.</td>
<td>1943-00012</td>
<td>&nbsp;</td>
<tr>
<td>Minimum Essential Media (MEM)</td>
<td>&nbsp;</td>
<td>Invitrogen (Gibco)</td>
<td>11090-081</td>
<td>with Earle's salts
without L-glutamine, needed for growing non-neuronal cultures</td>
</tr>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen (Gibco)</td>
<td>15070-063</td>
<td>for non-neuronal culture</td>
</tr>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Invitrogen (Gibco)</td>
<td>16140-071</td>
<td>needed for non-neuronal cultures</td>
</tr>
<tr>
<td>Nunclon "Triple Flask" Tissue Culture Bottle</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>12-565-25</td>
<td>Use for non-neuronal cultures. these flasks are very convenient when producing "conditioned Neurobasal Media/B27", but any other tissue culture flasks or dishes can be used instead.</td>
</tr>
<tr>
<td>Glass bottom "Imaging dishes"</td>
<td>&nbsp;</td>
<td>MatTek Corp.</td>
<td>P35G-1.5-10-C</td>
<td>Glass bottomed, &#216;35mm culture dishes ideal for Calcium or Sodium Imaging when using an inverted imaging setup. But expensive!</td>
</tr>		</tbody>
		</table>
		</div>

preparation of dissociated mouse cortical neuron cultures

此影片将引导的过程，产生胚胎后期和产后早期小鼠大脑的皮层神经元的文化。这些文化可用于各种应用，包括免疫细胞化学，生物化学，电生理，钙，钠成像，蛋白质和/或RNA隔离。这些文化也提供了一个平台，进行胚胎后期或出生后致死的基因突变的转基因动物研究神经发育。这个过程是相对直线前进，需要在组织培养技术的一些经验，不应该超过两到三个小时，如果你是正确准备。从丘脑 - 皮层纤维束的皮质外皮仔细分离，将减少不必要的非神经元细胞的数量。为了提高神经细胞的产量皮层组织碎片轻轻磨碎后酶的孵化步骤。这是必要的，因为它可以防止不必要的损伤细胞和神经细胞过早死亡。由于这些文化都保持在神经胶质细胞饲养层细胞的情况下，他们还提供了一个增长中的神经元丰富的文化附加值优势。

Watch this Scientific Journal Video about Preparation of Dissociated Mouse Cortical Neuron Cultures at JoVE.com

Preparation of Dissociated Mouse Cortical Neuron Cultures

这个视频显示了从胚胎后期和产后早期小鼠皮层神经元的文化生成的过程。这些文化可用于免疫细胞化学，生物化学，电生理，钙和钠成像，并提供一个平台，携带产后的致死基因突变的转基因动物研究神经元发育。

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 ...

preparation-of-dissociated-mouse-cortical-neuron-cultures

Research

JoVE Journal

Biology

43.3K 次观看.  University of California, Irvine (UCI). 这个视频显示了从胚胎后期和产后早期小鼠皮层神经元的文化生成的过程。这些文化可用于免疫细胞化学，生物化学，电生理，钙和钠成像，并提供一个平台，携带产后的致死基因突变的转基因动物研究神经元发育。

视频: Preparation of Dissociated Mouse Cortical Neuron Cultures

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 ...

科研

生物学

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 ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

小学游离中脑多巴胺鼠类新生儿的细胞培养

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

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 ...

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons 

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the cerebellar neuronal types (granule cells, Purkinje cells and inhibitory interneurons), granule neurons are by far the most numerous and are the most abundant type of neurons in the mammalian brain. In rodents, cerebellar granule neurons are generated during the first two post-natal weeks from progenitor cells in the outermost layer of the cerebellar cortex, the external granule layer (EGL). The protocol presented here describes techniques to enrich and culture granule neurons and their progenitor cells from post-natal mouse cerebellum. We will describe procedures to obtain cultures of increasing purity3,4, which can be used to study the differentiation of proliferating progenitor cells into granule neurons5,6. Once the progenitor cells differentiate, the cultures also provide a homogenous population of granule neurons for experimental manipulation and characterization of phenomena such as synaptogenesis, glutamate receptor function7, interaction with other purified cerebellar cells8,9 or cell death7.

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons

Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse.

产后小鼠小脑颗粒神经元祖细胞和神经元的分离和培养

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the ...

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) ...

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

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 ...

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to immortalized cell lines. However, the post-mitotic nature of primary neurons prevents effective heterologous protein expression using common procedures such as electroporation or chemically-mediated transfection. Thus, other techniques must be employed in order to effectively express proteins in these non-dividing cells.
In this article, we describe the steps required to perform intranuclear injections of cDNA constructs into dissociated adult sympathetic neurons. This technique, which has been applied to different types of neurons, can successfully induce heterologous protein expression. The equipment essential for the microinjection procedure includes an inverted microscope to visualize cells, a glass injection pipet filled with cDNA solution that is connected to a N2(g) pressure delivery system, and a micromanipulator. The micromanipulator coordinates the injection motion of microinjection pipet with a brief pulse of pressurized N2 to eject cDNA solution from the pipet tip. 
This technique does not have the toxicity associated with many other transfection methods and enables multiple DNA constructs to be expressed at a consistent ratio. The low number of injected cells makes the microinjection procedure well suited for single cell studies such as electrophysiological recordings and optical imaging, but may not be ideal for biochemical assays that require a larger number of cells and higher transfection efficiencies. Although intranuclear microinjections require an investment of equipment and time, the ability to achieve high levels of heterologous protein expression in a physiologically relevant environment makes this technique a very useful tool to investigate protein function.

Direct intranuclear injection of cDNA is an effective transfection technique for post-mitotic cells. This method provides high levels of heterologous protein expression from single or multiple cDNA constructs and enables protein function to be studied in a physiologically relevant environment with a variety of single cell assays.

Methods Article

游离的小鼠皮层神经元文化的制备