条纹分析来研究蛋白质底物使用游离海马神经元的吸引力或令人反感的活动
Summary
Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2’s ability to repel cultured hippocampal neurons.
Abstract
Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.
Introduction
轴突导向是由新形成的神经元的神经系统1,2开发过程中发出轴突到其目标的过程。显影轴突在其前端称为生长锥携带一个高度能动结构。生长锥检测外线索导航轴突的路径。导向分子，如狭缝，脑信号蛋白，肝配蛋白和，可以吸引或取决于它们与轴突1,3,4合适受体和共同受体相互作用击退轴突。该激活受体信号传送到影响其轴突和生长锥的运动细胞骨架组织生长锥。
各种方法已被开发来评估诱和驱避剂分子的作用。化疗诱和驱虫剂可施用与梯度浓度的生长/培养基（ 例如 ，唐恩的腔室或μ幻灯片）由微-对5,6，在一个高度集中点ipette（ 例如 ，车削试验）7，或通过浴应用程序（ 例如 ，生长锥塌陷测定法）8,9均匀浓度。
其它方法包括条纹测定或微接触印刷（μCP），其中一个化学引诱或斥涂覆的板的表面上作为底物10-12。 Thestripe法最初是由朋霍费尔和他的同事开发了在1987年以分析小鸡眼膜-顶盖系统13地形测绘。原始方法需要真空系统的外壳蛋白上使用的条纹和啮合矩阵聚碳酸酯核孔膜。在以后的版本中，重组蛋白用窄缝硅矩阵14,15直接印刷的培养板的表面上在一个条纹图案。近日，各研究小组已成功地应用这个条纹检测到轴突导向分子的活动16-21分析。
<p class ="“jove_content”">在这里，我们提出详细的协议为措施分离的海马神经元轴突导向分子的吸引或排斥的条纹检测。值得注意的是，这种方法可以在最低限度地配备实验室设置被应用。对于该测定，是在使用硅基质的塑料培养皿具有90微米缝隙产生并涂覆有层粘连蛋白的荧光标记的底物和对照蛋白的交替条纹。在我们的示范，解离从E15.5小鼠海马神经元分别在交替纤连蛋白和跨膜蛋白2（FLRT2）富含亮氨酸和控制Fc蛋白21的重组胞外结构域的条纹进行培养。培养24小时后，无论是轴突和神经元的细胞体强烈从FLRT2条纹排斥。用抗TAU1抗体染色揭示〜神经元的90％被分布在与Fc包被的区域，相比于上FLRT2-Fc的〜10％，这表明FLRT2具有很强的推斥力功能海马神经元21。Protocol
Representative Results
Discussion
这个协议描述了使用重组蛋白质和分离的神经元从E15.5小鼠海马的条纹检测。该测定允许神经元的排斥力，吸引力，或中性反应于感兴趣的重组蛋白放置在条纹图案的有效观察。该协议的主要优点是用于产生条纹，其中蛋白质被直接印刷到塑料盘的表面上的简单的方法，相对于传统的方法，该方法需要特殊的基质，真空系统，和一个核孔膜20 21。为了使蛋白条带，标记的重组蛋白可通过一?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (23700412, 25122707 and 26670090 to S.Y.).
Materials
| 15 mL centrifuge tube | Violamo | 1-3500-01 | |
| 4% Paraformaldehyde (PFA) | Nacalai | 01954-85 | |
| Alexa Fluor 488 Goat anti-human IgG antibody | Thermo Scientific | A11013 | |
| Alexa Fluor 594 Donkey anti-mouse IgG antibody | Thermo Scientific | A-21203 | Dilution 1/500 |
| Anti-Tau1 antibody | Chemicon | MAB3420 | Dilution 1/200 |
| Antifade | Thermo Scientific | P7481 | Alternative mounting media may be used |
| B27 supplement | Thermo Scientific | 17504-044 | Dilution 1/50 |
| Bovine serum albumin | Sigma | 01-2030-2 | |
| Cell strainer 100 um | BD Falcon | 352360 | |
| Centrifugation machine | Kubota | 2410 | |
| Cover glass 18mmx18mm | Matsunami | 18×18 mm No. 1 | |
| DAKO pen | DAKO | S2002 | Alternative water-repellent pen may be used |
| Disposable scalpel | Feather | 2975#11 | |
| FBS | Thermo Scientific | 10437-028 | |
| Fluorecent microscope | Nikon | E600 | |
| Forceps No. 5 | Fine Science Tools | 11254-20 | |
| GlutaMAX | Thermo Scientific | 35050-061 | Dilution 1/200 |
| Hamilton Syringe | Hamilton | 805N | 22 gauge, 50 uL |
| HBSS | Thermo Scientific | 14170-112 | |
| Human IgG, Fc Fragment | Jackson | 009-000-008 | |
| Laminin | Thermo Scientific | 23017-015 | |
| Neurobasal | Thermo Scientific | 21103-049 | |
| Normal Donkey Serum | Jackson | 017-000-121 | |
| PBS | Nacalai | 14249-24 | |
| Penicillin-Streptomycin | Thermo Scientific | 15070-063 | Dilution 1/100 |
| Plastic culture dish, 60 mm | Thermo Scientific | 150288 | |
| Silicone Matrices | Available and purchasable from Prof. Martin Bastmeyer (bastmeyer@kit.edu) | ||
| Stereo Microscope | Olympus | SZ61 | |
| Tip, 1000 uL | Watson | 125-1000S | |
| Transparent sticky tape | Tesa | 57315 | Alternative sticky tape may be used |
| Triton X-100 | Sigma | T8787 | |
| Trypan blue, 0.4% | Bio-Rad | 145-0013 | |
| Trypsin/EDTA | Thermo Scientific | 25300-054 | |
| Culture medium | Neurobasal supplemented with B27, GlutaMAX and Penicillin-Streptomycin. |
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