Name: live-cell imaging of migrating cells expressing fluorescently-tagged proteins in a three-dimensional matrix
Uploaded: 2011-12-22T13:00:00
Description: Watch this Scientific Journal Video about Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix at JoVE.com

我们感谢手稿的关键读博士格兰特隅田川。这项工作是由一个贝克曼青年研究员奖“（SY），赫尔曼家庭新教师奖”（SY），美国国立卫生研究院尤里卡，加州大学癌症研究中心协调委员会的支持。

1。稳定细胞株的产生（如MDCK细胞） <ol><li>板细胞在80-90％汇合在P35菜。不要让细胞形成100％汇合单层，这将降低转染效率。 </li><li>细胞转染质粒用脂质体2000的利益。优化转染条件下，使用制造商的协议。 </li><li>第二天，通过到两个P150皮氏培养皿中的细胞。大盘是建议允许足够的间距之间的稳定的殖民地。 </li><li>第二天，改变媒体和500微克/毫升的G418添加到每一道菜。 G418浓度应优化个别的细...

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	<li>Shih, W., Yamada, S., Myosin, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dependent+retrograde+flow+drives+3D+cell+migration.">A dependent retrograde flow drives 3D cell migration.</a> Biophys. J. 98 (8), L29-L31 (2010).</li><li>O'Brien, L. E., Yu, W., Tang, K., Jou, T. S., Zegers, M. M., Mostov, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+and+biochemical+analysis+of+Rac1+in+three-dimensional+epithelial+cell+cultures.">Morphological and biochemical analysis of Rac1 in three-dimensional epithelial cell cultures.</a> Methods Enzymol. 406, 676-691 (2006).</li><li>Legant, W. R., Miller, J. S., Blakely, B. L., Cohen, D. M., Genin, G. M., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+mechanical+tractions+exerted+by+cells+in+three-dimensional+matrices.">Measurement of mechanical tractions exerted by cells in three-dimensional matrices.</a> Nat. Methods. 7 (12), 969-971 (2010).</li><li>Del Alamo, J. C., Meili, R., Alonso-Latorre, B., Rodriguez-Rodriguez, J., Aliseda, A., Firtel, R. A., Lasheras, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatio-temporal+analysis+of+eurkaryotic+cell+motility+by+improved+force+cytometry.">Spatio-temporal analysis of eurkaryotic cell motility by improved force cytometry.</a> Proc. Natl. Acad. Sci. U.S.A. 104 (33), 13343-13348 (2007).</li><li>Lippincott-Schwartz, J., Snapp, E., Kenworthy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+protein+dynamics+in+living+cells.">Studying protein dynamics in living cells.</a> Nat. Rev. Mol. Cell Biol. 2 (6), 444-456 (2001).</li><li>Phair, R. D., Misteli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+modeling+approaches+to+in+vivo+imaging.">Kinetic modeling approaches to in vivo imaging.</a> Nat. Rev. Mol. Cell Biol. 2 (12), 898-907 (2001).</li><li>Giepmans, B. N., Adams, S. R., Ellisman, M. H., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+toolbox+for+assessing+protein+location+and+function.">Fluorescent toolbox for assessing protein location and function.</a> Science. 312 (5771), 217-224 (2006).</li><li>Shaner, N. C., Steinbach, P. A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+choosing+fluorescent+proteins.">A guide to choosing fluorescent proteins.</a> Nat. Methods. 2, 905-909 (2005).</li><li>Cukierman, E., Pankov, R., Stevens, D. R., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taking+cell-matrix+adhesions+to+the+third+dimension.">Taking cell-matrix adhesions to the third dimension.</a> Science. 294 (5547), 1708-1712 (2001).</li></ol>

在这里，我们描述了一个用活细胞成像研究细胞迁移的机制，在一个三维矩阵的方法。这项技术的成功取决于获得“良好”的克隆稳定表达GFP标记的蛋白质。绿色荧光蛋白的水平低会损害细胞的健康，而过高GFP水平将有不良的副作用，对细胞需要一个多余的激励曝光。因此，转染到细胞中的基因的载体选择是很重要的（例如，子，荧光标记等）。有很多绿色荧光蛋白，包括红色荧光蛋白（RFP?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>胶原蛋白，牛，I型</td><td> BD公司</td><td> 354231 </td><td>股票约3毫克/毫升</td></tr><tr><td> 3，aminopropyltrimethoxysilane </td><td>西格玛爱秩序</td><td> 281778 </td><td>稀释水</td></tr><tr><td>戊二醛</td><td>西格玛爱秩序</td><td> 340855 </td><td>在PBS稀释</td></tr><tr><td> 1M HEPES </td><td> Invitrogen公司</td><td> 15630-080 </td><td></td></tr><tr><td> Fluospheres聚苯乙烯微球1微米，红色荧光（六百〇五分之五百八十零） </td><td> Invitrogen公司</td><td> F13083 </td><td></td></tr><tr><td> Geneticin（G418） </td><td> Invitrogen公司</td><td> 11811-031 </td><td></td></TR&gt; <tr><td colspan="4"> 文化传媒组件： </td></tr><tr><td> DMEM培养液</td><td> Invitrogen公司</td><td> 31600-034 </td><td></td></tr><tr><td>胎牛血清</td><td>亚特兰大生物</td><td> S115500 </td><td></td></tr><tr><td>青霉素/链霉素</td><td> Invitrogen公司</td><td> 15140-122 </td><td></td></tr><tr><td>卡那霉素</td><td> Invitrogen公司</td><td> 15160-054 </td><td></td></tr></tbody></table>

live-cell imaging of migrating cells expressing fluorescently-tagged proteins in a three-dimensional matrix

传统上，细胞迁移进行了研究二维的，僵硬的塑料表面上。然而，在伤口愈合，组织再生和癌症转移，如重要的生物过程，细胞必须通过复杂的，立体化的细胞外组织的导航。为了更好地理解这些生物过程背后的机制，重要的是要研究负责驱动细胞迁移的蛋白质的作用。在这里，我们勾勒出一个协议研究使用的上皮细胞株（MDCK）细胞迁移的机制，和一个立体的，纤维，作为一个模型系统的自我聚合矩阵。这透明的细胞外基质是活细胞成像研究容易服从和更好地模拟生理，软组织环境。这份报告展示了一个直接可视化蛋白定位和动态的技术，和周围的三维矩阵​​变形。 考试蛋白定位在细胞过程的动态ination到蛋白质的功能提供了关键的洞察力。基因编码的荧光标记提供了一个独特的方法观察蛋白定位和动态。使用这种技术，我们可以分析细胞内积累的关键，强行通过基质细胞演习实时生成细胞骨架的组成部分。此外，使用多个不同波长的荧光标记，我们可以研究多种蛋白质同时进行本地化，从而让我们来测试，例如，不同的蛋白质是否具有类似或不同的角色。此外，荧光标记的蛋白质的动态，可量化的使用荧光漂白后（FRAP）分析恢复。这种测量方法检测蛋白的流动性和稳定约束的蛋白质是细胞骨架网络。 通过与蛋白质功能抑制剂治疗相结合的活细胞成像，我们可以检查实时的蛋白质和细胞迁移形态的分布变化。此外，我们还结合使用荧光​​示踪粒子矩阵内的嵌入式可视化在细胞迁移的矩阵变形的活细胞成像。因此，我们可以想像如何迁移细胞分布力产生的蛋白质，以及牵引力施加到周围基质。通过这些技术，我们可以得到宝贵的见解，成特定的蛋白质和他们的贡献细胞迁移机制的角色。

Watch this Scientific Journal Video about Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix at JoVE.com

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

传统的细胞过程，如细胞迁移研究二维的，僵硬的塑料表面上。本报告介绍了一个直接可视化蛋白定位和分析一个有关生理，立体矩阵的细胞迁移的蛋白质动力学的技术。

活细胞成像的移植细胞在一个三维矩阵表示荧光标记的蛋白质

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

Research

JoVE Journal

Bioengineering

Three-dimensional Optical-resolution Photoacoustic Microscopy

三维光学分辨率的光声显微镜

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

科研

生物工程

Planar and Three-Dimensional Printing of Conductive Inks

平面和立体印刷的导电油墨

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

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Analysis of Cell Migration within a Three-dimensional Collagen Matrix

在一个三维胶原蛋白矩阵分析细胞迁移

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

使用Cell-基板阻抗和活细胞成像，以衡量矩阵修改诱导实时变化的细胞粘附和去粘附

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

三维全层皮肤的生成等效和自动伤人

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Live Cell Imaging during Mechanical Stretch

在机械拉伸活细胞成像

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

工程三维上皮组织细胞外基质中嵌入

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

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This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

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A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...