从Midgastrula舞台果蝇胚胎神经文化的制备
Summary
这个视频演示midgastrula阶段果蝇胚胎的主要神经文化的准备。生活文化的意见表明电镀和后2天在碳酸氢盐为基础的定义中等的增长有区别的神经元细胞1小时后。神经元电兴奋，并形成突触连接。
Abstract
该视频演示了midgastrula阶段果蝇胚胎的主要神经文化的过程。收集胚胎和dechorionation使用漂白水的方法证明。我们说明了一嘴吸痰管使用玻璃吸管，从单一胚胎细胞清除。分散成小（5升）下降中等未涂层的玻璃盖玻片上的每一个embyro细胞的方法是证明。一个电镀后1小时，通过显微镜说明首选的细胞密度。大多数细胞的生存时，在定义中等增长是神经母细胞分裂前12-24小时延长神经炎进程的一个或多个次文化。通过显微镜说明突起生长的水平，预计在2天的体外分支在一个健康的文化。的文化是生长在一个简单的碳酸氢钠为主的定义介质在5％二氧化碳培养箱中培养，在22-24 ° C。神经炎进程继续阐述文化的第一个星期，当他们从邻近的细胞，他们往往形成功能性突触连接的突起联系。在这些文化中的神经元表达电压门控钠，钙，钾通道，电兴奋。这种文化系统是用于研究分子遗传和环境因素，调节神经细胞的分化，兴奋性突触的形成/功能。
Protocol
Discussion
在果蝇的文化准备midgastrula阶段的胚胎的神经元产生的，其中许多鸿沟之前，在体外分化成神经细胞前体。因此，该系统提供了一个独特的机会，在神经元发育的早期阶段（Rohrbaugh等，2003）中的重要探索遗传和环境因素。我们已经表明，生长在一个简单的定义介质的神经元分化成神经元电兴奋，也形成功能性突触连接（直径多德，1995年李和O多德，1999年）。使用分析突变体和/或药理操作，在这个模型系统可用于…
Acknowledgements
这项工作是由美国国立卫生研究院授予NS27501 DKOD支持。这从霍华德休斯医学研究所教授格兰特DKOD工作的额外支持。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Drosophila melanogaster | Animal | Fruit flies | ||
| Transferrin | Reagent | Sigma | T-1147 | 100x Stock: 10 mg/ml in water. Filter through 0.2 um syringe filter (cellulose acetate). Store in 220 ul aliquots (for 20 ml DMEM) at -20C. |
| Insulin | Sigma | I-6634 | 200x Stock: 10 mg/ml in 0.05N HCl. Filter through 0.2 um syringe filter (cellulose acetate). Store in 120 ul aliquots (for 20 ml DMEM) at -20C. | |
| Putrescine | Sigma | P-5780 | 100x Stock: 10 mM in ddH2O. Filter through 0.2 um syringe filter (cellulose acetate). Store in 220 ul aliquots (for 20 ml DMEM) at -20 C | |
| Selenium | Sigma | S-5261 | 100x Stock: 3 uM in ddH2O. Put 0.0051 g Selenium in a 15 ml tube labeled A (3 mM stock). Add 10 ml of sterile water. Take 10 ul from Tube A to Tube B with 10 ml ddH2O (3 uM stock). Filter Tube B through 0.2 um syringe filter (cellulose acetate). Store in 220 ul aliquots (for 20 ml DMEM) at -20C | |
| Progesterone | Sigma | P-6149 | 100x Stock: 2 ug/ml 1. Add 1ml of 100% EtOH to 0.001 g Progesterone bottle 2. Add 49 ml ddH2O 3. Transfer 1 ml of this to second tube with 9 ml ddH2O 4. Filter through 0.2 um syringe filter (cellulose acetate) 5. Store in 220 ul aliquots (for 20 ml DMEM) at -20C | |
| DDM1 | Medium | To 10 mls of DMEM add from stocks: 100 ul Transferrin, 100 ul Putrescine, 100 ul Selenium, 100 ul Progesterone, 50 ul Insulin | ||
| Petri dishes | ||||
| Cover-slips | Bellco Biological Glassware | 1943-00012 | Low lead glass, autoclaved. | |
| Paper filter | Whatman | #1 | ||
| 10cc syringe | Tool |
The cultures made in this media must be maintained in a 5% CO2 incubator at about 22-24°C. We use a standard mammalian tissue culture incubator placed in a cold room and heated to 23°C. Always used autoclaved filtered water and make in containers that are reserved for media and supplements only. Never put a pH meter or stir bar used for other purposes in media.
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