分离和游离感觉神经元的文化活动从鸡胚
Summary
细胞培养模型提供了环境条件的详细控制，从而提供一个强大的平台，以阐明神经元细胞生物学的许多方面。我们描述了一种快速，廉价和可靠的方法来从鸡胚分离，解离和培养的感觉神经元。还提供基质制剂和免疫细胞化学的细节。
Abstract
神经元是多层面的细胞中携带信息的各种功能，包括感觉，运动运动，学习和记忆至关重要。 体内研究神经元可以是具有挑战性的，因为它们的复杂性，其变化的动态环境，和技术的限制。由于这些原因， 在体外研究神经元可以证明是有益的解开神经元的复杂的奥秘。的细胞培养模型中良好定义的性质提供了对环境条件和变量的具体控制。在这里，我们介绍了如何分离，分解，并从鸡胚培养初级神经元。这种技术是快速，廉价，并且产生强劲增长的感觉神经元。该过程持续产生培养物被高度富集的神经元，并具有很少的非神经元细胞（小于5％）。原代神经元没有很好地附着在未经处理的玻璃或组织培养塑料，因此详细的程序来创建两个迪斯丁CT，定义良好的含有层粘连蛋白基质为神经镀进行说明。培养的神经元是高度适合于多种细胞和分子生物学技术，包括免疫共沉淀，活细胞的想象，RNA干扰，和免疫细胞化学实验。关于这些培养的神经元的双免疫程序进行了优化，并在此描述。
Introduction
神经元是复杂的细胞传递信息的各种功能，包括感觉，视觉，运动运动，学习，记忆至关重要。从其他细胞类型的独特，神经延长臂状突起，称为轴突，以形成必要的神经公路进行通信。在位于轴突生长的秘诀开发专门车厢，称为生长锥，浏览外线索的音乐会导致轴突到相应的目的地。背后生长锥的导航复杂的分子机制尚不完全清楚。为了更好地理解这些机制，研究人员已经用细胞培养模型，研究了定义，并简化在体外环境中的神经元。在文化学习1神经元，导致了我们的神经元细胞生物学的理解，包括显著进展：神经细胞的分化2，细胞骨架动力学，细胞内吞作用和贩运，枝晶规3,4，轴突再生5，和临床病症，如神经病6。此外，培养的神经元是高度适合于范围广泛的研究的技术，包括免疫细胞化学，细胞表面的共免疫沉淀，Western印迹，转染，RNA干扰，和实时成像等的生长锥运动缩时分析。因此，培养初级神经元是一种有效的方法来阐明神经元的细胞生物学的许多方面。
细胞培养模型提供与调查员对环境条件和变量详细的控制。例如，在其上的神经元被镀（并且在生长）的基质可以容易地操纵。这里，我们提供了用于产生两个不同的基质，一种具有低的层粘连蛋白-1的浓度和其它具有饱和的层粘连蛋白-1的浓度的详细说明。出人意料的是，不同浓度的相同的分子可具有显着影响对神经元的内部状态，以及它们的细胞表面成分。例如，整合的cAMP和表面水平的细胞内水平是在神经细胞铺于两个基质7,8显著不同。另外的研究表明，其它分子，包括纤连蛋白和硫酸软骨素蛋白多糖，影响细胞表面分子的表达和神经元运动7-11。此外，可溶性分子如神经营养因子和neurotropins也影响细胞膜的组成和神经运动12-16并且可以在细胞培养模型中很容易地和准确地操作。
在这里，我们描述的方法来分离和培养解离来自鸡胚的感觉神经元。这个过程已经被用来制造在神经生物学显著突破，包括轴突生长5,7,8,10,11,16-21和从设计到分离神经节细胞22中的程序进行了修改。有几个优点这种方法。首先，鸡背根神经节（DRG）开发的许多功能都得到很好的特点，包括出生，轴突延长的时间框架和蛋白质表达谱2,23-28，从而提供了赖以建立翔实的体外实验的指导性依据。第二，解离的神经元培养物使研究者能够更直接地学习的神经元相比，使用含有两个游离的神经元和非神经元细胞完好的DRG外植体（其包含神经元和非神经元细胞）和/或混合培养物的替代方法。第三，这里描述的方法很简单，价格低廉，适合于大学生。因此，这种技术可以用于研究以及用于教学目的。此外，该协议的微小变化应该让神经元并非来自病种付费等速度快，产量高的净化。例如，这个过程可以被修改以提供neuronally ENR从其它组织iched培养如胚胎前脑或脊髓。
免疫细胞化学的协议已经被优化用于这些分离的神经元培养物和这里进行了详细的说明。提供了一种用于对神经细胞粘附分子（NCAM）和β1整合素双重免疫细胞的步骤。从这些免疫细胞化学方法产生的数据已被用于检查空间构图和几个分子的强度在培养的神经元8,16。
Protocol
1，盖玻片准备：酸洗和烘烤至少提前2天清扫（步骤2），开始下面的步骤准备盖玻片。进行酸洗步骤以去除油脂，彻底清洁的盖玻片。 负载盖玻片瓷支架的垂直位置盖玻片，防止它们相互接触。淹没支架和盖玻片放入盛有2M的盐酸（HCl）的玻璃容器中组织学。或者，如果瓷支架是无法使用，散布〜20盖玻片水平地覆盖100毫米的玻璃陪替氏培养皿的底部，并浸没在2 M盐酸盖玻片。 …
Representative Results
这里所描述的协议使研究者培养解离胚胎感觉神经元具有很少（ 例如 ，<5％）非神经元细胞7,8,10,16的富集群体。许多病种付费可从腰骶部，胸椎和颈椎区域获取。根据研究者的需要，从这些不同的解剖区域的DRG可以容易地分离。例如， 图1示出了鸡胚通过夹层与腰椎的DRG不同阶段的图像在图1C和图1D和图1E和1F胸椎的DR…
Discussion
在这里，我们提出了详细的协议进行分离培养分离从鸡胚感觉神经元。这个过程产生强劲增长的神经元的体外 7,8,10,16富集群体。众多的细胞和分子生物学技术可以应用到这些培养的神经元中，包括免疫细胞化学，在此所说明。该协议是最近使用的定量评估免疫标记激活整合素在感官生长锥8,16力度。在这些先前的研究中，软件程序被用于准确地创建了生长锥的顶端20微?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们要感谢艾莉森菲尔布鲁克，贝琳达Barbagallo和迈克尔·弗朗西斯对本文有见地的意见。研究报告本出版物是由R15区奖1R15NS070172-01A1授予MLL支持。
Materials
| sterile small culture dishes (35mm) | Corning | 430165 | |
| sterile large culture dishes (100mm) | Falcon | 353003 | |
| sterile large petri dishes (100mm) | VWR | 89000-302 | |
| glass petri dish (100mm) | VWR | D108962 | |
| fine forceps | Fine Science Tools | 11251-10 | |
| standard forceps | Fine Science Tools | 1100-12 | |
| coverslips (22×22) | Fisher | 12 518 105K | 0.13-0.17 mm thickness is optimal for microscopy |
| porcelin coverslip holder | Thomas scientific | 8542E40 | |
| Fetal Bovine serum | Gibco | 17502-048 | use 50ml aliquots per 500 mls of Ham's F12 media |
| HCL | VWR | VW3204-1 | use at 2M, can be used twice before discarding |
| NaCl | Sigma | S9888 | use 5M NaCl solution for laminin-1 stock solution |
| laminin-1 | Invitrogen | 23017-015 | Add 72ul of sterile 5M NaCl and aliquot into 50ul |
| 15 ml conical tube | cell treat | 229411 | |
| PBS CMF 10X | Gibco | 14200 | used at 1X, dilute with sterile millipore filtered water |
| PBS 10X | Gibco | 14080 | used at 1X, dilute with sterile milliporee filtered water |
| Ham's F12 | Lonza | 12-615F | |
| Fetal Bovine Serum Albumin (BSA) | Gibco | 10438 | use in F12HS20 at 10% |
| HEPES | Sigma | H3375 | make sterile 1M solution in distilled water, dilute in F12 media to obtain final concentration of 10mM |
| Penicillin streptomyocin | Sigma | P0781 | use 5mls in 500mls of F12 media |
| NT3 | Millipore | GF031 | Add 1ml of sterile millipore water, aliquot under sterile conditions, store at -20 0C, use at final concentration of 10ng/ml in F12H media, keep in -20 C non-defrosting freezer |
| N2 | Invitrogen | 17502048 | aliquot under sterile conditions,stored at -20 0C, use 100ul per 10ml of F12H media |
| NGF | RnD systems | 256-GF | Add 1ml of sterile 1X PBS with 0.1% BSA, aliquot under sterile conditions, store at -20 0C, usd at final concentration of 10 ng/ml in F12H media |
| l-glutamine | Sigma | G7513 | aliquot under sterile conditions, store at -20 0C, use at final concentration of 200mM in F12H media |
| Trypsin | Sigma | T4049 | aliquot under sterile conditions, store at -20 0C |
| Bovine Serum Albumin (BSA) | Gibco | 15260 | |
| Other items needed: general dissection instruments, including glass pasteur pipettes, fertile white leghorn chicken eggs, check egg incubator (humidified, 37 degrees C), cell incubation chamber (humidified, 37 degrees C), laminar flow hood, binocular stereovision dissecting scope | |||
| Immunocytochemistry Reagents Table | |||
| Sucrose | Sigma | S9378 | used in fixative solution (4% paraformaldehye, 30% sucrose, 2X PBS) |
| Triton-X 100 | Sigma | T-8787 | |
| Paraformaldehyde | Sigma | P6148 | used in fixative solution (4% paraformaldehye, 30% sucrose, 2X PBS) |
| Fluoromount G | Southern Biotech | 0100-01 | |
| normal goat serum | Life technologies | PCN500 | |
| microscope slides | VWR | 16004-430 | |
| Primary Antibody Table | |||
| Antibody against NCAM | Millipore | AB5032 | polyclonal |
| Antibody against activated beta 1 ingegrin | Millipore | MAB19294 | monoclonal |
| Seconday Antibody Table | |||
| Goat anti-mouse IgG Alexa 488 | Life technologies | A11001 | |
| Goat anti-rabbit IgG Alexa 548 | Life technologies | A11036 |
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Citar este artigo
Powell, S., Vinod, A., Lemons, M. L. Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos. J. Vis. Exp. (91), e51991, doi:10.3791/51991 (2014).