基质贴壁细胞的微管吸吮的估计单元刚度
Summary
在这里，我们描述了一个快速和简单的方法来测量细胞的刚度。这种方法的一般原则是响应于施加到细胞表面通过微量吸移管的良好定义的负压来测量膜变形。这种方法提供了一个功能强大的工具，研究基板贴壁细胞的生物力学特性。
Abstract
越来越多的研究表明，在多种细胞功能，包括细胞增殖，分化，迁移和细胞间的相互作用，单个细胞的生物力学性能中扮演重要角色。细胞生物力学的两个关键参数是细胞的变形能力，刚度和能力的细胞收缩，产生力量。在这里，我们描述了一种快速和简单的方法来估计细胞通过测量在由玻璃微到细胞表面，被称为微吸管或Microaspiration一种技术，响应于施加的负压的程度的膜变形的刚度。
通过拉动玻璃毛细管创建一个微量吸移管，用一个非常小的尖端（2-50微米直径的细胞或组织样本的大小取决于），然后将其连接到一个气动压力换能器，并带来了一个密切进行microaspiration周边的细胞在显微镜下观察。当的移液管的尖端接触的细胞，负压的工序，被施加到吸液管的充气产生良好定义的细胞膜上的压力的压力传感器。以响应于压力，膜被吸入到吸液管和渐进的的膜变形或进入吸移管被作为时间的函数的测量的“膜投影”。本实验的方法的基本原则是，响应于定义的机械力的膜的变形程度是一个函数的膜刚度。更硬的膜，膜变形和率越慢越短的稳态的抽吸length.The技术可以执行分离的细胞，无论是在悬浮液和基板连接，大的细胞器，和脂质体。
进行分析，通过比较不同的细胞群或实验条件下，一个给定的压力下实现最大的膜的变形。 A“刚性系数”是EStimated通过绘制送气膜变形长度作为所施加的压力的函数。此外，可以进一步分析数据来估计细胞的杨氏模量（E），最常见的参数来表征的材料的刚度。重要的是要注意，可以被看作是真核细胞的质膜，其中深层次的由子膜细胞骨架，它是细胞骨架构成的膜的机械支架，占主导地位的可变形膜的脂质双层的双组分系统的蜂窝的信封。这种做法，因此，可以探测子膜细胞骨架的生物力学特性。
Protocol
1。拉玻璃微 设备：：微管拔轮器，Microforge。 玻璃：Boroscillicate玻璃毛细管（〜1.5毫米的外部直径，〜1.4毫米的内直径）。 微量被拉到使用相同的基本方法，是用于制备玻璃微电极电生理录音。简要地说，在中间加热的玻璃毛细管，和当玻璃开始熔融时的两个半体的毛细管被拉开产生两个微量。多个商业的…
Discussion
microaspiration提供了一种简单和高重现性的方法来估计细胞的刚度/可变形性，通过施加负压力至细胞膜和测量细胞膜变形响应于良好定义的压力。它最早是由Mitchison和斯旺（1954年）的海胆卵的弹性性能特征的精辟见解的机制，细胞分裂21，然后看在红血细胞的机械性能。这种方法已被用于多项研究评估的生物力学特性不同的细胞类型（ 如 2,3,12,13）。我们最近的研究扩展了?…
Disclosures
The authors have nothing to disclose.
Materials
| Name of reagent | Company | Catalog/Model Number | Comments |
| Sutter pipette puller | Sutter Instruments | P-97 | |
| Microforge | Narishige | MF-830 | |
| Inverted Fluorescent Microscope | Zeiss | Axiovert 200M | The microscope should be preferably equipped with 3D/deconvolution capabilities. |
| Videocamera | Zeiss | AxioCam MRm | |
| Image Acquisition sotware | Zeiss | AxioVision | |
| Pneumatic Pressure Transducer | BioTek | DPM-1B | DPM1B Pneumatic Transducer Tester can now be found by FLUKE. |
| Pipette glass | Richland | Customized glass | Pipettes were customized with a 1.2 inner diameter and 1.6 outer diameter. |
| DiI Dye | Invitrogen | D282 | Dissolves well in DMSO |
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Tags
Micropipette AspirationSubstrate-attached CellsCell StiffnessBiomechanical PropertiesCellular DeformabilityCellular StiffnessCell ContractionForce GenerationMembrane DeformationNegative PressureGlass MicropipetteMicropipette Aspiration TechniqueMicroaspirationGlass CapillaryPneumatic Pressure TransducerMembrane ProjectionExperimental Approach
Cite This Article
Oh, M., Kuhr, F., Byfield, F., Levitan, I. Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness. J. Vis. Exp. (67), e3886, doi:10.3791/3886 (2012).