在体外细胞迁移和侵袭实验
Summary
常用的，非常方便的方法研究细胞迁移和侵袭的体外描述。第一种方法是测量细胞运动的细胞伤口愈合测定法。第二种方法是，评估的趋化和细胞的侵袭能力Transwell小迁移和侵袭实验。
Abstract
Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.
Introduction
蠕动是活细胞的一个重要特征。细胞迁移是参与生命，胚胎发育，免疫反应，以及许多病理过程，如癌症转移和炎症1-9的概念。因此，方法来研究细胞的迁徙行为是非常有用的研究工具，在生物医学科学，生物学，生物工程及相关领域学科门类齐全的。
在癌症研究中的细胞迁移的研究是特别感兴趣，因为死亡的癌症患者的主要原因是关系到转移进程。为了让癌细胞扩散和传播遍及全身，癌细胞必须迁移和入侵，通过细胞外基质（ECM），intravasate进入血液循环，连接到一个远程站点，最后渗出，形成远处病灶1,10〜12。各种生物的方法可以用来详细研究这些事件。细胞培养伤口闭合的D中的Transwell小迁移和入侵检测被广泛应用于科学界1,10。这些测试可以提供必要的数据，可能会允许对如何以及特定细胞类型可以自发地迁移或响应一个化疗诱和定向朝向其迁移的理解。几个迁徙的表型进行了说明。细胞可以迁移见于间质或变形虫样运动或标记的集体迁移或细胞流13多细胞运动的单细胞形式如。在游动细胞用于运动的方法，可以使用细胞培养物的伤口闭合测定法可以容易地观察到。
在众多的方法来研究细胞迁移，细胞伤口缝合法是最简单的一种。这种方法可用于确定整个细胞团的迁移能力。时采取的一个步骤进一步它可以用于迁移14时，观察各个细胞的形态特征。随着伤口闭合的分析很多表型可能显露出来。比较控件时测量近距离随着时间的推移可能会发现特定迁移量的变化或受损迁徙的表型是前所未知的。此外，单细胞lamellipodium形成，尾部回缩，并定向运动可能会给什么可能会削弱或增强的利息14细胞的线索。
Transwell小迁移和侵袭测定法可用于分析单个细胞的定向到不同的化疗诱响应它们是否是趋化因子，生长因子，脂质，或核苷酸4,5,8,15,16的能力。它也可以评估差分迁移能力因过度表达的受体1,14的。这些测定法还可以用于鉴定和表征细胞迁移的关键调节剂，如Rho家族的小GTP酶2。下面的这些短且容易存取权限sible测试中，细胞迁移的模式和小区的侵入成三维基质的能力也可以被确定。
Protocol
Representative Results
Discussion
细胞迁移是在癌症研究，研究的一个重要方面，它也可以应用到发育，免疫和伤口愈合研究。细胞培养伤口闭合试验和Transwell小细胞迁移和侵袭测定法揭示了细胞迁移性的行为的详细信息，并且可以用于研究细胞迁移1,2,10,14的分子机制。我们的研究中使用这些细胞运动性测定法来确定B16F10黑色素瘤细胞系的迁移速度和侵袭能力。
细胞伤口愈合试验检查一个特定细胞系…
Declarações
The authors have nothing to disclose.
Acknowledgements
The authors thank Mike Myles, Sam Saunders, and C.W. Elton at the ECU Multimedia & Technology Services for providing assistance in video production. We acknowledge the grant support from North Carolina Biotechnology Center, Golfers against Cancer, Brody Brothers Endowment Fund, American Heart Association, and ECU/Vidant Cancer Research and Education Fund (L.V.Y. and M.J.R.).
Materials
| Dulbecco’s modified Eagle medium (DMEM) | Gibco | 11995-073 | |
| Fetal bovine serum (FBS) | Gemini | 100106 | |
| Bovine serum albumin (BSA) | Sigma-Aldrich | A4503-50G | |
| Trypsin EDTA 0.25% | Gibco | 25200-056 | |
| Dulbecco’s phosphate buffered saline (DPBS) | Gibco | 14190-250 | |
| Crystal violet | Sigma | C0775-100G | Dissolved in water at 0.2% |
| Cell disassociation buffer | Gibco | 13151-014 | |
| Cell culture incubator | Thermo Fisher Scientific | Model # 3145 | |
| SterilGARD biosafety hood | The Baker Company, Inc. | Model # VBM-600 | |
| EVOS Fl inverted microscope | Thermo Fisher Scientific | Model # AMF-4302-US | |
| Tissue culture plate | Becton Dickinson | 353046 | Catalog number varies depending on the type of culture plate |
| Corning Transwell insert | Fisher | 07-200-150 | Catalog number varies depending on the pore size of the membrane |
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