躯干神经嵴细胞迁移使用一个修改过的Zigmond室法分析
Summary
取出的细胞迁移（躯干神经嵴细胞）的方法来分析。这种方法是廉价的，温柔的，和能够区分趋化学增活现象和其他因素的影响，如在主躯干神经嵴细胞培养的细胞 – 细胞相互作用所产生的迁徙极性的。
Abstract
神经嵴细胞（NCCS）是一个流动人口的细胞存在于脊椎动物的发展，移民从背神经管（NT）后发生上皮-间质转化1,2。在EMT，NCC的长距离迁移，沿刻板的途径，直到他们达到他们的目标。 NCC的区分成为一个巨大的数组类型的细胞，包括神经元，神经胶质细胞，黑素细胞和嗜铬细胞1-3。 NCC的接触和认识到自己的正确的目标位置的能力是基本适当的主干NCC-源性成分结构的形成。因此，阐明机制的指导主干NCC迁移的问题具有十分重要的意义。大量的分子已被证明来指导NCC迁移4。例如，躯干NCC的Semaphorin，和ephrin和缝配体的5-8负指导线索，如被击退。然而，并非直到最近，已确定9的任何趋化因子的主干NCC的。
常规的体外方法研究的贴壁细胞的趋化行为的工作最好与永生化的，均匀分布的细胞，但更具挑战性的适用于某些初级最初缺乏均匀的分布和迅速分化（如NCC的）的干细胞培养。均匀化分布的主干NCC的趋化研究的一种方法是从小学NT植文化的主干NCC的隔离，然后抬起replate他们几乎100％汇合。然而，这种电镀的方法需要大量的时间和精力，到取出足够的细胞，是残酷的，分布躯干的NCC的在一个不同的方式，发现在体内条件。
在这里，我们报道了在体外的方法，是可以评估的趋化和其他迁徙的主干NCC的回应没有requirin嘎同质细胞分布。此技术利用延时成像伯，泰然自若改性Zigmond室（一个标准Zigmond室其他地方描述10）内的中继线净捐助国。通过暴露在培养的周边的中继线净捐助国到趋化梯度是垂直于他们的预测的自然方向性，由所施加的趋化梯度诱导洄游极性的改变可以被检测。这种技术是廉价的，需要只有两个NT外植体每重复处理的培养，避免恶劣的细胞起重（如胰蛋白酶消化），叶中继线NCC的在一个更相似的分布，在体内条件下，外植和实验之间的时间量减少（这可能会降低分化的风险），并允许时间的推移众多的候鸟特性的评价。
Protocol
Discussion
在主干NCC的趋化研究已经证明了具有挑战性的一系列原因。主干的NCC的构成一个异构干细胞的人口将分化，如果培养长期的，因此，躯干NCC的，必须从主外植的中继级NT。传统的方法来研究均匀分布在体外的细胞群的趋化反应是难以在主干NCC的测试，因为他们首先需要的细胞是孤立的均匀再接种的趋室（例如，Boyden小室12）。为外植几十个NTS可能会被要求创建一个均匀分布的主干NCC?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们特别感谢利诺金，史蒂夫古兹曼和Ujit的Satyarthi的，这种方法在开发过程中的技术援助。迈伦·霍桑，理查德·Spengel，罗伯托·罗哈斯加工室使用，提供了必要的技术援助。值得一提的是，罗伯托·罗哈斯图4。我们也感谢斯科特·弗雷泽发展的趋化试验前的宝贵意见。这项工作得到部分支持，NIH-MBRS分数5S06GM048680-13 MEDB和加州州立大学北岭分校研究生论文支持计划奖CW。
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| DMEM | Omega Scientific | DM-22 | |
| Penicillin Streptomycin Solution | Omega Scientific | PS-20 | 100X Stock Concentration |
| L-Glutamine | Omega Scientific | GS-60 | 100X Stock Concentration |
| Fetal Bovine Serum | Omega Scientific | FB-11 | Lot# 105247 (or another that is comparable) |
| Modified Zigmond chamber | Home made | N/A | Reservoir volume: ~ 160 μl ea; for additional specifications, see Fig. 4 and the supplemental fabrication protocol |
| Cell culture dish | Denville | T6040 | 40 x 10 mm |
| Fibronectin | BD | 354008 | 10X Stock prepped by diluting 1 mg FN in 1 ml H2O and 9 ml DMEM |
| Coverslips | Fisher | 12-548-B | Precleaned; 22 x 22 mm |
| L15 medium | Thermo Scientific | SH30525.02 | |
| Petroleum Jelly | Comforts | 011110794642 | 100% |
| Centrifuge tube | Biologix | 10-9152 | 15 ml |
| Dispase | Cell Systems | 4Z0-850 | 10X Stock Concentration |
| Syringe | BD | 309602 | 1 ml |
| Needle | BD | 305127 | 25 G x 1.5 in. |
| Alexa Fluor 488-IgM | Invitrogen | A21042 | Stock is 2 mg/ml; 7 moles dye/mole IgM |
| Dissecting Forceps | FST | Misc. | Dumont #5 or 55; straight tipped; stainless steel or titanium |
| Tungsten Needle | N/A | N/A | Home made; placed in a pin holder |
| Blunt Forceps | Tiemann | 160-18 | Used for transferring embryos to Ringer’s from egg yolk |
Supplemental Protocol: Fabrication of a Modified Zigmond Chamber
Please refer to Figure 4 as a reference for the protocol below:
- Purchase a sheet of 3/16″ thick polished acrylic (4.45 mm actual thickness).
- Using a table saw, cut chamber blanks oversized to the rough dimensions of 33.25 mm x 64.57 mm. This allows 3.175 mm extra material for machining.
- Set the chamber blank on a vise. With a milling machine and a 6.35 mm (1/4″) end mill bit, finish machining the sides of the chamber to their exact dimensions: 30.07 mm x 61.39 mm.
- Position the chamber blank on the milling machine and locate the center of the blank along both the x and y axes with an edge finder; then zero the center location.
- Acquire the chamber height (z-axis) by touching the end mill bit to the top surface and zero the height.
- Using a 3.91 mm (0.154″) end mill bit, offset the bit 3.03 mm along the x-axis (positive direction) for the first reservoir. Begin machining into the chamber to a depth of 2.84 mm while moving along the y-axis (positive direction) to 7.62 mm (0.300″) and then traverse to 7.62 mm (0.300″) in the opposite (negative) direction to a complete reservoir length of 15.24 mm (0.600″). Offset the bit to 3.03 mm (0.119″) along the x-axis (negative direction) and repeat the same process for the second reservoir.
- Position the chamber on its edge and drill a hole using a 1.09 mm (0.043 in.) drill bit on the end of each reservoir (4 total) that connects the end of the reservoir to the side of the chamber for loading medium during experimentation.
- Soak the chamber well in warm soapy water to help remove any chemical contaminants.
- Soak and rinse the chamber well in double-distilled water to remove any soap. The chambers are now ready to use as described above.
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