Name: a simplified system for evaluating cell mechanosensing and durotaxis in vitro
Uploaded: 2015-08-27T15:00:00.000+00:00
Duration: 9 min 50 s
Description: Watch this Scientific Journal Video about A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro at JoVE.com

这项工作是由美国国立卫生研究院R01 GM47607，CA163296和NSF 1334493至CET支持。我们感谢特纳实验室成员的手稿批判性阅读。本报告中显示的所有数据均来自Wormer 等2014 11转载许可。

1.准备Durotaxis商会<ol><li>为了制备1 6-孔组织培养板，去皮用50毫升锥形管中的平衡。称取约10克，在50毫升试管中的PDMS碱溶液中（该溶液是非常粘稠）。 </li><li>对于90:1的衬底（符合性〜1千帕），通过90分在管与PDMS碱溶液的测定重量来确定的交联剂溶液所需的正确的金额。与PDMS交联剂溶液的计算量添加到同一管中。 例如:10克/ 90 = 0.11克。添加0.11克PDMS交联剂溶液与PDMS碱溶液。 注意:基地和交联剂溶液在试剂盒的PDMS都提供。 </li><li>大力5分钟在RT使用小刮勺混合PDMS基/交联剂混合物。在这个阶段，混合物将含有大量气泡。 </li><li>离心机在台式离心机5分钟的PDMS衬底在50×g离心在RT以除去泡泡糖BLES。在5分钟，离心后重新如果仍有气泡。 </li><li>吸管1毫升90:1的PDMS衬底进入组织培养的每孔处理的6孔板中。存在于PDMS任何残留的气泡可以在本阶段通过使用21g的针弹出它们被消除。允许PDMS散布在井30分钟。 </li><li>煮沸12个毫米的玻璃＃1盖玻片在蒸馏水中5分钟。重复两次，并存储在蒸馏水盖玻片。 </li><li>放置一个，干燥盖玻片进入组织培养板的各孔中，轻轻触摸盖玻片一侧插入的PDMS溶液然后删除盖玻片上的PDMS。作为盖玻片沉降，与PDMS将开始侵占过盖玻片边缘，但不会完全覆盖它。这将产生固化后的PDMS和玻璃之间的界面（见图1A 中的B）。 </li><li>孵育板在70℃的烘箱中16小时以固化（硬化）的PDMS。放置高原E在细胞培养罩和紫外线消毒10分钟。 注意:最好是在使用了几天做出板。然而，板可以包在石蜡膜而没有任何缓冲，并储存在4℃达2周没有任何质量明显下降。 </li></ol> 2.细胞电镀注意:如果学习siRNA-介导击倒的效果，执行使用制造商的说明或所选择的细胞类型的优化的协议的击倒。 <ol><li>涂层的每个durotaxis室用1毫升10微克/毫升纤连蛋白在PBS无钙和镁的1小时，在37℃。替代地，纤连蛋白可以在天在4℃下施加在分析前通过将与PDMS用10微克/毫升纤连蛋白在PBS中16小时。确保整个表面浸没在在PBS溶液中的纤连蛋白。 </li><li>制备热变性1％BSA中。称取0.5克BSA，溶于50毫升的PBS。过滤消毒通过0.22微米的过滤器和热的溶液，在80℃下12分钟。 注意:准备该溶液与细胞电镀和储存在4℃下之前的一天。 </li><li>吸出纤连蛋白溶液和洗涤3次，用PBS洗涤。在PBS中添加1 ml热变性BSA的每个孔中，温育30分钟，在室温。 </li><li> Trypsinize和计数选择的细胞，而durotaxis室阻挡的热变性BSA。 </li><li>板1×10 5个细胞在一体积为2ml进入durotaxis室的每个孔中，使用所需的选择的特定细胞类型的介质。使细胞附着并在培养箱散布在基板上4小时，在37℃，5％的CO 2。 注:U2OS细胞例行维持在含10％FBS补充了2mM L-谷氨酰胺，1mM丙酮酸钠，和10国际单位/毫升青霉素，10微克/毫升链霉素。 注意:在本实施例中所用的细胞数目为优化U2OS细胞。可能需要优化的其他类型的细胞。该密度使细胞足够的空间，而无需与其他细胞相互作用显著迁移。 </li></ol> 3.活细胞成像<ol><li>在倒置显微镜下进行活细胞成像，使用了10X物镜相衬。该显微镜应装有一个封闭的环境，加湿室，允许温度的期间长期成像控制，在37℃和5％的CO 2。 </li><li>在细胞已经扩散约3.5小时，组装板显微镜室。使样品（多个）在腔室中平衡30分钟。 </li><li>设置自动，多点来访显微镜上（如果可用）。聚焦90之间的界面上:1的PDMS和玻璃盖玻片并选择各地，平均每durotaxis室40点的接口点图像。图像将细胞每10分钟达16小时。 注:重感兴趣只园将显示为两行以对应于盖玻片边缘和对应于PDMS和盖玻片之间的实际接口的内线路的外线路。参见图1B。 </li></ol> 4.数据分析<ol><li>生成的电子表格，如表1。 </li><li>算的交叉事件从PDMS从生成的每个电影的玻璃表面，反之亦然数。记录在Excel电子表格相应列的交叉事件的数量。 注意:交叉事件被定义为细胞核越过PDMS和玻璃在任一方向的边界。 </li><li>量化多个道口，计数细胞越过界面的次数。该号码应记录在对应于在其上电池位于电影结束基板excel的列。重复分析每一个跨越接口在T细胞他的电影。排除出视场的成像期间迁移的细胞。 注:同样重要的是，以验证，通过细胞在实验开始时算起，该细胞能够同样附着在纤连蛋白涂覆的PDMS和玻璃盖玻片表面。 </li><li>计算细胞的从PDMS迁移到玻璃表面（即，经历durotaxis）的百分比。添加渡从软到硬且倍数交叉事件，通过交叉事件的总数结束了硬和分裂事件的数目。 </li><li>通过将多个横跨的数目由交叉的总数计算的多个交叉的百分比。 </li></ol>

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The authors have no conflicts to disclose.

在此我们描述了一个简单的实验来研究durotaxis在细胞迁移。此法的主要优势是易于使用准备PDMS中durotaxis室的。基片的刚性可以通过改变碱的PDMS溶液的比率交联剂，以允许在测定中的各种刚性的研究很容易被操纵。然而，该系统中的一个潜在的限制是，细胞仅暴露于基板的刚性的单一变化，而 ​​不是经历由更复杂的系统4提供一个刚性梯度。重要的是，不象聚丙烯酰胺基质中，ECM组分不?...

细胞外基质（ECM）是由结构和交联蛋白，包括胶原蛋白，纤连蛋白和层粘连蛋白的一系列复杂的。虽然它是公认的对ECM提供了细胞组织重要的结构支承，有越来越多的证据表明，细胞积极应对物理变化在其细胞外基质环境来调节多种细胞过程，包括细胞存活，分化和细胞迁移。例如，在ECM的刚性差异能驱动间充质干细胞向不同谱系，具有柔软基材（〜1千帕）促进神经谱系而僵硬（〜25千帕）衬底促进成骨分化1。同样地，增加间质基质刚性已经显示出促进乳腺上皮细胞肿瘤的发生和侵袭到周围组织2,3。 这mechanosign的一个特别有趣的方面阿玲活性导致称为durotaxis，其中细胞优先迁移朝着更刚性基板4,5的处理。细胞不断通过整联蛋白受体感知它们的胞外环境的物理特性结合到ECM。这，反过来，促使许多结构性和信号蛋白的积累到其胞质结构域，以驱动被称为粘着斑或局灶性触点6,7-粘合剂结构的形成。 自整联没有固有的酶活性，信号是从ECM中继通过这些辅助蛋白来协调细胞的反应其变化的环境8。因此，参与调节mechanosignaling和durotaxis关键蛋白质的鉴定和表征是调查的一个重要领域。 各种模型系统已被开发，研究durotaxis 体外 ，但最有利用胶原包被的聚丙烯酰胺基质4。然而，聚丙烯酰胺基片的制备中可以是技术上具有挑战性并在这些试验中使用的胶原必须是化学交联到基底9。聚二甲基硅氧烷（PDMS）基片已被证明表现出的聚丙烯酰胺基片10可比机械性能。然而，PDMS衬底被通过简单地混合碱的比率来制备交联剂和这些基片可以涂有ECM蛋白无需化学交联，从而使PDMS一个更简单的工具，研究刚性对细胞行为的影响。在此，我们介绍如何编写一个简单durotaxis室，其中软PDMS基板集成了硬质玻璃盖玻片。 该法，如下所述，为研究durotaxis一种快速，简便的方法。在这项研究中，我们使用人类U2OS骨肉瘤细胞结合的siRNA介导cdGAP击倒研究在durotaxis 11本黏着蛋白的作用。重要的是，该协议可以很容易地适应个人的要求。其它细胞类型可被取代的U2OS细胞和任何蛋白质可以被击倒或过表达，以确定在durotaxis对细胞行为的影响。此外，该协议可以适于掺入荧光标记的蛋白来分析他们的动态和用FRAP行为或FRET方法。

<table><tbody><tr><td>Polydimethylsiloxane (PDMS)</td><td>Dow Corning</td><td>3097358-1004</td><td>Sylgard 184 Silicone Elastomer Kit</td></tr><tr><td>#1 Cover glass 12 mm</td><td>Fisher Scientific</td><td>12-545-82</td><td></td></tr><tr><td>6-well plate</td><td>Celltreat</td><td>229106</td><td></td></tr><tr><td>DMEM</td><td>Cellgro</td><td>15-017-CM</td><td></td></tr><tr><td>L-Glutamine</td><td>Cellgro</td><td>25-005-CI</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Fisher Scientific</td><td>BP356-100</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Cellgro</td><td>30-002-CI</td><td></td></tr><tr><td>Fibronectin</td><td>BD Biosciences</td><td>610077</td><td></td></tr><tr><td>PBS</td><td>Invitrogen</td><td>21600-044</td><td></td></tr><tr><td>Falcon tubes</td><td>Celltreat</td><td>229456</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Atlanta Biologicals</td><td>S11150</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>U2OS cells</td><td>ATCC</td><td>HTB-96</td><td></td></tr></tbody></table>

a simplified system for evaluating cell mechanosensing and durotaxis in vitro

细胞外基质的成分和机械性能是组织类型之间高度可变。此结缔组织基质多样性大大影响细胞的行为，以调节正常和病理过程，包括细胞增殖，分化，粘附信令和定向迁移。在这方面，某些细胞类型的固有能力，以移向称为durotaxis较硬的，或更少的标准矩阵衬底。这种现象扮演胚胎发育，伤口修复和肿瘤细胞入侵期间起重要作用。这里，我们描述一个简单的试验来研究durotaxis， 在体外，用聚二甲基硅氧烷（PDMS）的基板。的描述durotaxis腔室准备创建相对软的PDMS凝胶和刚性玻璃盖玻片之间的刚性的接口。在提供的例子中，我们已经使用了这些durotaxis室展示为CDC42作用/ Rac1的GTP酶激活prote在，cdGAP，在mechanosensing和人类U2OS骨肉瘤细胞durotaxis调节。该测定是易于适应其它类型的细胞和/或利益的其他蛋白质的敲低，探讨在mechanosignaling和durotaxis各自的作用。

所述durotaxis室的示意图示于图1A。软PDMS衬底（90:1的混合物的PDMS碱与交联剂溶液）被铺在6孔皿，玻璃盖玻片放置在PDMS，然后部分地覆盖盖玻片的上表面的顶部上，从而产生一个接口不同的顺应的两个基板之间。软PDMS基板的刚性是〜1千帕，这与脑组织的典型合规性，而玻璃的刚性是大约1-2 GPA 1。所述PDMS和玻璃之间的界面的相衬图像显示由白色箭头在图1B。星号表?...

Watch this Scientific Journal Video about A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro at JoVE.com

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

为评估细胞Mechanosensing和Durotaxis简化系统<em&gt;体外</em

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity ...

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Biology

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

8.2K 次观看.  SUNY Upstate Medical University. Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

视频: A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to understand morphogenetic processes is to directly measure cellular forces and mechanical properties in vivo during embryogenesis. Here, we present a setup of optical tweezers coupled to a light sheet microscope, which allows to directly apply forces on cell-cell contacts of the early Drosophila embryo, while imaging at a speed of several frames per second. This technique has the advantage that it does not require the injection of beads into the embryo, usually used as intermediate probes on which optical forces are exerted. We detail step by step the implementation of the setup, and propose tools to extract mechanical information from the experiments. By monitoring the displacements of cell-cell contacts in real time, one can perform tension measurements and investigate cell contacts&#39; rheology.

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

We present a setup of optical tweezers coupled to a light sheet microscope, and its implementation to probe cell mechanics without beads in the Drosophila embryo.

无珠光高音喇叭在食子胚胎中的细胞力学探测

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to ...

科研

发育生物学

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila larvae, customized metal nickel-titanium alloy (nitinol) filaments. These mechanical probes are similar to the von Frey filaments used in vertebrates to measure mechanical nociception. Here, we demonstrate how to make and calibrate these mechanical probes and how to generate a full behavioral dose-response from subthreshold (innocuous or non-noxious range) to suprathreshold (low to high noxious range) stimuli. To demonstrate the utility of the probes, we investigated tissue damage-induced hypersensitivity in Drosophila larvae. Mechanical allodynia (hypersensitivity to a normally innocuous mechanical stimulus) and hyperalgesia (exaggerated responsiveness to a noxious mechanical stimulus) have not yet been established in Drosophila larvae. Using mechanical probes that are normally innocuous or probes that typically elicit an aversive behavior, we found that Drosophila larvae develop mechanical hypersensitization (both allodynia and hyperalgesia) after tissue damage. Thus, the mechanical probes and assay that we illustrate here will likely be important tools to dissect the fundamental molecular/genetic mechanisms of mechanical hypersensitivity.

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

The goal of this protocol is to show how to perform an improved assay for mechanical nociception in Drosophila larvae. We use the assay here to demonstrate that mechanical hypersensitivity (allodynia and hyperalgesia) exists in Drosophila larvae.

测量 果蝇 拉瓦的机械结节的改进的检测和工具

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila ...

行为学

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

机械性刺激，集成光学和原子力显微镜研究活细胞的反应

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

生物学

Direct Force Measurements of Subcellular Mechanics in Confinement using Optical Tweezers

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue morphogenesis goes in hand with molecular and structural changes at the single cell level that result in variations of subcellular mechanical properties. As a consequence, even within the same cell, different organelles and compartments resist differently to mechanical stresses; and mechanotransduction pathways can actively regulate their mechanical properties. The ability of a cell to adapt to the microenvironment of the tissue niche thus is in part due to the ability to sense and respond to mechanical stresses. We recently proposed a new mechanosensation paradigm in which nuclear deformation and positioning enables a cell to gauge the physical 3D environment and endows the cell with a sense of proprioception to decode changes in cell shape. In this article, we describe a new method to measure the forces and material properties that shape the cell nucleus inside living cells, exemplified on adherent cells and mechanically confined cells. The measurements can be performed non-invasively with optical traps inside cells, and the forces are directly accessible through calibration-free detection of light momentum. This allows measuring the mechanics of the nucleus independently from cell surface deformations and allowing dissection of exteroceptive and interoceptive mechanotransduction pathways. Importantly, the trapping experiment can be combined with optical microscopy to investigate the cellular response and subcellular dynamics using fluorescence imaging of the cytoskeleton, calcium ions, or nuclear morphology. The presented method is straightforward to apply, compatible with commercial solutions for force measurements, and can easily be extended to investigate the mechanics of other subcellular compartments, e.g., mitochondria, stress-fibers, and endosomes.

Here, we present a protocol to investigate the intracellular mechanical properties of isolated embryonic zebrafish cells in three-dimensional confinement with direct force measurement by an optical trap.

使用光学镊子测量禁闭中的亚细胞力学的直接力

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

一个行为分析对机械感觉MARCM为基础的克隆<em&gt;果蝇</em

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

神经科学

Measurement of Cellular Chemotaxis with ECIS/Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted 1. In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues 2.
Another assay system, the under-agarose chemotaxis assay, 3, 4 measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays.
The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) 5, 6. In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system 6. In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. 
Dictyostelium discoidium is known to migrate in the presence of a folate gradient 7, 8 and its chemotactic response can be accurately measured by ECIS/Taxis 9. Leukocyte chemotaxis, in response to SDF1&alpha; and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1&alpha; is shown in Figure 1.

The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.

ECIS的/出租车细胞趋化测量

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

Mechano-Node-Pore Sensing: A Rapid, Label-Free Platform for Multi-Parameter Single-Cell Viscoelastic Measurements

Cellular mechanical properties are involved in a wide variety of biological processes and diseases, ranging from stem cell differentiation to cancer metastasis. Conventional methods for measuring these properties, such as atomic force microscopy (AFM) and micropipette aspiration (MA), capture rich information, reflecting a cell's full viscoelastic response; however, these methods are limited by very low throughput. High-throughput approaches, such as real-time deformability cytometry (RT-DC), can only measure limited mechanical information, as they are often restricted to single-parameter readouts that only reflect a cell's elastic properties. In contrast to these methods, mechano-node-pore sensing (mechano-NPS) is a flexible, label-free microfluidic platform that bridges the gap in achieving multi-parameter viscoelastic measurements of a cell with moderate throughput. A direct current (DC) measurement is used to monitor cells as they transit a microfluidic channel, tracking their size and velocity before, during, and after they are forced through a narrow constriction. This information (i.e., size and velocity) is used to quantify each cell's transverse deformation, resistance to deformation, and recovery from deformation. In general, this electronics-based microfluidic platform provides multiple viscoelastic cell properties, and thus a more complete picture of a cell's mechanical state. Because it requires minimal sample preparation, utilizes a straightforward electronic measurement (in contrast to a high-speed camera), and takes advantage of standard soft lithography fabrication, the implementation of this platform is simple, accessible, and adaptable to downstream analysis. This platform's flexibility, utility, and sensitivity have provided unique mechanical information on a diverse range of cells, with the potential for many more applications in basic science and clinical diagnostics.

Presented here is a method to mechanically phenotype single cells using an electronics-based microfluidic platform called mechano-node-pore sensing (mechano-NPS). This platform maintains moderate throughput of 1-10 cells/s while measuring both the elastic and viscous biophysical properties of cells.

机械节点孔传感:用于多参数单细胞粘弹性测量的快速、无标记平台

Cellular mechanical properties are involved in a wide variety of biological processes and diseases, ranging from stem cell differentiation to cancer ...

生物工程

Mechanostimulation of Multicellular Organisms Through a High-Throughput Microfluidic Compression System

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such as aspiration or coverslip compression have been used to mechanically stimulate whole embryos. These approaches limit experimental design as they are imprecise, require manual handling, and can process only a couple of embryos simultaneously. Microfluidic systems have great potential for automating such experimental tasks while increasing throughput and precision. This article describes a microfluidic system developed to precisely compress whole Drosophila melanogaster (fruit fly) embryos. This system features microchannels with pneumatically actuated deformable sidewalls and enables embryo alignment, immobilization, compression, and post-stimulation collection. By parallelizing these microchannels into seven lanes, steady or dynamic compression patterns can be applied to hundreds of Drosophila embryos simultaneously. Fabricating this system on a glass coverslip facilitates the simultaneous mechanical stimulation and imaging of samples with high-resolution microscopes. Moreover, the utilization of biocompatible materials, like PDMS, and the ability to flow fluid through the system make this device capable of long-term experiments with media-dependent samples. This approach also eliminates the requirement for manual mounting which mechanically stresses samples. Furthermore, the ability to quickly collect samples from the microchannels enables post-stimulation analyses, including -omics assays which require large sample numbers unattainable using traditional mechanical stimulation approaches. The geometry of this system is readily scalable to different biological systems, enabling numerous fields to benefit from the functional features described herein including high sample throughput, mechanical stimulation or immobilization, and automated alignment.

The present protocol describes the design, fabrication, and characterization of a microfluidic system capable of aligning, immobilizing, and precisely compressing hundreds of Drosophila melanogaster embryos with minimal user intervention. This system enables high-resolution imaging and recovery of samples for post-stimulation analysis and can be scaled to accommodate other multicellular biological systems.

通过高通量微流体压缩系统对多细胞生物进行机械刺激

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such ...

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both force-sensing molecules at adhesion sites, and force-dependent transcription factors that regulate lineage-specific gene expression and drive phenotypic outputs. However, the signaling networks converting mechanical tension into biochemical pathways have remained elusive. To explore the signaling pathways engaged upon mechanical tension applied to cell surface receptor, superparamagnetic microbeads can be used. Here we present a protocol for using magnetic beads to apply forces to cell surface adhesion proteins. Using this approach, it is possible to investigate not only force-dependent cytoplasmic signaling pathways by various biochemical approaches, but also adhesion remodeling by magnetic isolation of adhesion complexes attached to the ligand-coated beads. This protocol includes the preparation of ligand-coated superparamagnetic beads, and the application of define tensile forces followed by biochemical analyses. Additionally, we provide a representative sample of data demonstrating that tension applied to integrin-based adhesion triggers adhesion remodeling and alters protein tyrosine phosphorylation.

Cell surface adhesions are central in mechanotransduction, as they transmit mechanical tension and initiate the signaling pathways involved in tissue homeostasis and development. Here, we present a protocol for dissecting the biochemical pathways that are activated in response to tension, using ligand-coated magnetic microbeads and force application to adhesion receptors.

分析细胞表面粘附重塑回应机械张力使用磁珠

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

对急性机械刺激的网柄反应的评估

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...

为评估细胞Mechanosensing和Durotaxis简化系统

摘要

探索更多视频

此视频中的章节

无珠光高音喇叭在食子胚胎中的细胞力学探测

测量果蝇拉瓦的机械结节的改进的检测和工具

机械性刺激，集成光学和原子力显微镜研究活细胞的反应

使用光学镊子测量禁闭中的亚细胞力学的直接力

一个行为分析对机械感觉MARCM为基础的克隆

ECIS的/出租车细胞趋化测量

机械节点孔传感:用于多参数单细胞粘弹性测量的快速、无标记平台

通过高通量微流体压缩系统对多细胞生物进行机械刺激

分析细胞表面粘附重塑回应机械张力使用磁珠

对急性机械刺激的网柄反应的评估

为评估细胞Mechanosensing和Durotaxis简化系统

摘要

探索更多视频

此视频中的章节

无珠光高音喇叭在食子胚胎中的细胞力学探测

测量 果蝇 拉瓦的机械结节的改进的检测和工具

机械性刺激，集成光学和原子力显微镜研究活细胞的反应

使用光学镊子测量禁闭中的亚细胞力学的直接力

一个行为分析对机械感觉MARCM为基础的克隆

ECIS的/出租车细胞趋化测量

机械节点孔传感:用于多参数单细胞粘弹性测量的快速、无标记平台

通过高通量微流体压缩系统对多细胞生物进行机械刺激

分析细胞表面粘附重塑回应机械张力使用磁珠

对急性机械刺激的网柄反应的评估

测量果蝇拉瓦的机械结节的改进的检测和工具