机械性刺激，集成光学和原子力显微镜研究活细胞的反应
Summary
本文旨在指导读者在原子操作的综合力量光学成像显微镜文化的活细胞的机械性刺激。一个一步一步的协议。一个有代表性的数据集显示活细胞的机械性刺激的反应。
Abstract
要理解这一机制的活细胞检测的机械力，以及他们如何应对和适应他们的环境，新的技术能够在亚细胞水平研究细胞的行为，具有高时空分辨率的开发。集成与全内反射荧光（TIRF）显微镜和快速旋转的磁盘（FSD）的共聚焦显微镜，原子力显微镜（AFM）。大致横跨在任何附着的活细胞的分子动力学研究的一个广泛适用的集成系统，可以直接实时的机械刺激细胞反应的光学成像。微丝和粘着斑的重大重排的结果表明，由于地方根尖细胞表面的机械刺激，诱导到整个细胞体细胞结构的变化。这些创新技术将提供新的信息，了解机械力活细胞的结构调整和动态。一个详细的协议和有代表性的数据显示活细胞机械性刺激的反应是。
Protocol
1。集成的显微镜系统概述这些研究中使用的显微镜系统，已被描述详细1。简单地说，一个倒置的奥林巴斯IX – 81 TIRF附件显微镜（奥林巴斯，中心谷，PA）结合CSU – 22横河扫描头（横河电机公司，日本）。一个放映机深圳原子力显微镜（威科仪器公司，圣巴巴拉，CA）是安装在光学倒置显微镜上，和整个系统的研究级光学表上方（新港，IRVINE，CA）。 <p class="jove_content"…
Discussion
细胞骨架的 7-10和粘着斑 11-16是动态的结构组合，分散和细胞迁移或回应机械力 17 。他们有重要的功能，调节细胞生长，生存，和基因表达。知道他们的具体分配和动态，提供了一个更好地了解细胞如何与基体和它们之间的沟通。
这种新的显微镜系统是大致跨越任何附着的活细胞的分子动力学研究的广泛适用。集成的显微镜，使亚细胞快速变化的定?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
纽- GFP质粒午山田，美国国立卫生研究院，国立牙科和颅面研究所，马里兰州贝塞斯达，和肌动蛋白- MRFP的质粒的礼物，是一个礼物，佛罗里达州立大学，佛罗里达州塔拉哈西，从迈克尔戴维森。
这项工作是支持由美国国家科学基金会职业奖＃0747334和AHA国家SDG＃0835205N Andreea气管。
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| AFM tip | Novascan Technologies | 2 micron Borosilicate bead, 0.01N/m, Biotin Surface | ||
| avidin | Sigma Aldrich | A9275 | 1mg/mL in DPBS | |
| Fibronectin | Sigma Aldrich | F4759 | 1 mg/mL | |
| DPBS | Invitrogen | 21600-051 | ||
| Amaxa transfection kit for Smooth Muscle Cells | Lonza | VPI-1004 | ||
| Mattek glass bottom dishes | MatTek Corporation | P60G-1.5-30-F | Sterile 60mm Falcon dishes with glass bottom coverslip no. 1.5 |
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Tags
Live Cell ResponseMechanical StimulationIntegrated Optical MicroscopyAtomic Force MicroscopySub-cellular LevelSpatial ResolutionTemporal ResolutionTotal Internal Reflection Fluorescence MicroscopyFast-spinning Disk Confocal MicroscopyMolecular Dynamic StudiesAdherent Live CellsOptical ImagingReal-timeActin FilamentsFocal AdhesionsCellular StructureCell RestructuringDynamicsDetailed Protocol
Citar este artículo
Trache, A., Lim, S. Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy. J. Vis. Exp. (44), e2072, doi:10.3791/2072 (2010).