CG是由来自法新社法国国家（ANR-13-JSV1-0008），来自欧盟第七框架计划（居里夫人职业集成n˚8304162），由根据欧盟的地平线欧洲研究委员会（ERC）资助项目2020年研究和创新计划（ERC启动格兰特n˚639300）。

1.配体偶联磁珠注意:使用超顺磁性甲苯磺酰基活化的珠用直径2.8微米，进行配体缀合（原液浓度10 8珠/毫升，30毫克珠粒/毫升）。以下协议是基于约2×10 5个细胞，这对应于在一个60mm组织培养板上生长至80％汇合MRC-5细胞的样品。相应地，如果使用不同大小或细胞的板在不同汇流调整的珠粒和试剂的体积。使用顺序的超顺磁珠的量具有每单元2珠。因此，需要用于60毫米板4×10 5珠。 <ol><li>通过涡旋至少30秒彻底重新悬浮在原始小瓶的珠子。等分试样40微升的再悬浮超顺磁珠（相应于4×10 6珠）至1.5毫升离心管中。 </li><li> 放置在管上的磁性separati上支架以将磁珠从溶液中分离出来。弃去上清液，从磁性分离底座上卸下管和在1mL的0.1M磷酸钠pH 7.4的重悬珠子。 <ol><li>用1mL的0.1M磷酸钠pH 7.4的重复洗涤步骤两次。 </li></ol></li><li>最后一次洗涤后，重悬珠子于1mL的0.1M磷酸钠pH 7.4的。 </li><li>结合100微克牛纤连蛋白（FN）或任何其他细胞粘附受体配体以含有小珠的1毫升的0.1M磷酸钠pH 7.4的并通过移液混合。 </li><li>通过更换FN或用BSA，聚-D-赖氨酸或脱辅基转铁蛋白为阴性对照的任何其他特异性配体重复前面的步骤。 </li><li>任选地，用于凝胶分析，采取的配位体溶液中的10μL的等分试样对的交联效率分析，用浓的Laemmli样品缓冲液的适当体积混合。 </li><li>孵育为12-24小时，在37℃上的转子的配位体含有溶液珠。 </li><li>如果小珠的聚集体出现的反应发生，声处理（连续39功率W）不超过10-20之后一下。 </li><li>隔离用磁选支架上的珠子，吸出剩余的溶液，并添加1毫升的PBS / 0.2％BSA的pH 7.6的溶液。孵育在37℃下于转子1小时。 </li><li>用1毫升的PBS / 0.2％BSA的pH值7.6洗使用磁体珠两次。悬浮珠粒于1mL的PBS / 0.2％BSA的pH值7.6。 </li><li>超声处理珠子如果出现聚集不超过20-30秒。 </li><li>或者，删除10微升等分来分析免疫印迹耦合效率（用浓Laemmli样品缓冲液适当体积混合）。 </li><li>继续在4℃下对细胞测定或商店珠粒长达1个月。 </li><li>任选地，运行SDS-PAGE凝胶和染色用考马斯蓝给FN交联分析到磁性珠粒。 </li></ol>的张力对绑定到粘附受体在背配体涂层珠2.应用细胞表面<ol><li>在适当的生长培养基60mm组织培养皿培养贴壁细胞（通常的DMEM为4.5g / l D同时葡萄糖补充有FCS的10％），直至达到80％汇合。 </li><li>制备非变性非离子裂解缓冲液（20mM的Tris盐酸pH值7.6，150 mM氯化钠，2mM的MgCl 2的，0.1％的NP-40）和冷却的离心管中。 </li><li>从颗粒部分1.10，重悬涂配体珠1毫升的PBS / 0.2％BSA pH值7.6。添加涂布配体珠粒溶液至5毫升温暖的培养基和旋涡的100微升。 </li><li>至60mm培养皿中吸并加入5毫升辅以珠温暖的生长介质。 </li><li>孵育细胞培养条件下20分钟（37℃和5％的CO 2），以允许珠粒沉淀并附着到细胞中。关键的是要不超过20分钟，因为珠可以通过吞噬作用被内化。 </li><li>或者，观察使用10X或20X OBJE光学显微镜下珠莫如和微微颤抖的菜连接并漂珠区分检查珠附着力。 </li><li>同时保持培养皿在培养箱，交换为一个盖子正常盘盖与安装在该上表面上的圆38毫米钕磁铁。磁体代替由定位在盖的下表面两个较小13毫米钕磁铁保持。由于磁铁是非常强大的，小心操作它们。 </li><li>孵育进行在细胞培养条件张力所需的时间点（37℃和5％CO 2）的细胞。 </li><li>治疗后，置于冰上菜，小心吸从盘整个网上平台。 </li><li>添加细胞裂解缓冲液的300微升，并孵育在冰上10分钟。收集用细胞刮刀，并转移到预冷的1.5毫升微量离心管中的溶胞产物。 </li><li>沉淀用磁选支架磁珠和总细胞裂解物转移到一个新的预寒意编1.5毫升离心管中。在-20℃下用于进一步分析存储此级分。 </li><li>用1mL冰冷的裂解缓冲液洗涤珠粒3次。添加50微升Laemmli样品缓冲液至珠粒料，使用干块加热器5分钟，在95℃，通过移液和煮沸混合。这部分包含了孤立的粘连物。继续生化分析或储存在-20°C。 </li><li>从后珠粒分离（约300微升）中获得的总细胞裂解物，除去免疫印迹分析50微升等分试样。 注:左，可用于进一步的生化研究，如蛋白质 - 蛋白质相互作用的研究（免疫沉淀，GST下拉测定）或GTP酶活性实验的250微升（改变裂解缓冲液可以根据所关注的GTP酶是必要的）。 </li></ol>

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作者宣称没有竞争经济利益。

此处所描述的方法构成，以施加张力到细胞表面粘附受体，并允许其随后的纯化一个简单的方法。然而，一些步骤是进行高效粘附纯化的和潜在的优化可以做取决于目标粘附受体的关键。我们目前的用户可能会遇到下面的潜在问题。 我们使用直径2.8微米的磁性珠粒但较大的小珠可用于直径如4.5微米。然而，珠子直径应限制在2-5微米自吞噬可能在30-60分钟温育发生更快速和更大...

在后生动物，机械张力通过细胞过程如增殖，分化和存活1,2无数的调节指导组织发育和内环境稳定。机械张力可以从细胞外基质产生，或可通过贴壁细胞，其样品通过拉动到细胞外基质，并通过张力敏感分子探测其刚性的肌动球蛋白收缩机械它们的胞外环境中产生的。为了应对紧张局势，机械敏感粘附蛋白发生触发复杂的信号级联反应的构象变化。反过来，这些信号传导途径编排一个mechanoresponse包含增殖，分化和存活是调节细胞行为到细胞外环境。这样的方法可以在短期时间周期进行结算（数秒至数分钟）快速反馈到机甲的环<html>通过修改机械敏感结构notransduction。例如，基于整联粘连加强在通过的Rho GTP酶-介导的细胞骨架重构3，4，5响应于张力。与此同时，其他的信号通路超过几小时或几天来控制的遗传程序，最终影响细胞的命运6激活。然而，许多研究强调矩阵刚度对细胞确定性和疾病发展1,2的效果，粘附介导的机械传导的精确分子机制仍然不清楚。 各种方法已被开发来研究细胞的行为的细胞产生的力或外力，包括流动系统，荧光共振能量转移（FRET）-tension传感器7的影响，小姑娘="外部参照"&gt; 8，柔性基板9，磁镊子，光学镊子10和原子力显微镜（AFM）11。在这里，我们使用超顺磁珠响应于施加到特定粘附受体的张力来表征机械传导途径提出的协议。超顺磁珠是放置在一个磁场时可逆地磁化的颗粒。一旦涂覆有对特定受体的配体，这些珠粒提供了有力的工具来研究细胞外力的应用程序的影响。此方法已被若干研究3，5，12验证- 17和呈现的优点在很大程度上促进在贴壁细胞的生化分析。使用类似的胶原包被的磁珠，随后通过生物化学分析，早期工作报告中的增加蛋白酪氨酸磷酸化和RhoA激活响应于张力5，18，19。下面描述的方法也被用于与纤连蛋白（FN）包被的珠从施加到整3张力下游表征信号通路。在这项研究中，Guilluy等。表明张力通过两个鸟嘌呤核苷酸交换因子（全环基金），LARG和全球环境基金-H1，以整合的粘附物招募激活的RhoA。因为，在其他的研究已经表明，GEF-H1被用不同的方法20，21，表现出这里描述的方法的稳健性招募到粘附物响应于细胞产生的张力。其结果是，活化的RhoA被证明促进粘附加固，通过细胞骨架重构。该系统也被用来探索张力施加吨O单元/细胞粘附受体。力到涂有诱导纽蛋白招募增加同样地整合伴生附着配合物12 E-钙粘蛋白的胞外域磁珠应用。柯林斯和他的同事观察到紧张的应用PECAM-1促进整合和RhoA的活性13。采用磁珠另一种实验方法是紧张适用于孤立的核研究。使用涂覆有对核包膜蛋白nesprin-1抗体珠，核膜复合物进行纯化，以显示它们响应于机械张力22动态地调节。这些结果支持在机械传导途径的研究该方法的有力度。此外，虽然流或牵引力系统刺激一般的细胞过程，磁珠特别通过使用受体配体靶向细胞粘附受体<sup系列="外部参照"&gt; 3或反对细胞表面受体13，15单克隆抗体。 这种方法的另一个优点是粘附复合物通过一个简单的配体的亲和纯化过程的隔离。这是众所周知的，加入的涂覆配体珠的细胞结合粘附受体和诱导几个粘附蛋白23的募集。力的涂覆配体磁珠进一步应用开启这些粘附配合成介导的各种张力依赖性信号通路4,24大分子平台。细胞裂解，随后通过使用磁铁珠浓度允许的附着平台的隔离。用于纯化粘附物的其它方法已经在贴壁细胞使用。他们结合化学交联，以节省蛋白质 - 蛋白质相互作用和细胞裂解步骤由洗涤剂和剪切流动或超声处理20，21，25，26，27，28。最后一步是含有粘合性复合物所得到的腹侧质膜的集合。不像这些方法，磁珠允许通过选择性靶向粘附受体的特定家族细胞粘附复合物的更大的纯化水平。磁珠已经用于纯化粘附络合物附着到涂覆配体的微珠29,30非贴壁细胞。下面模拟生物环境中描述的方法，其中施加短持续时间（数秒至数分钟）力。因此，它提供了一个有力的工具，调查两个纯化粘附复合物的分子组成和下游机械敏感-信号通路。 在这里，我们提出了一个详细的实验协议利用磁珠的张力适用于粘接面的蛋白质。永久钕磁铁被放置在培养皿表面的顶部。磁铁的磁极面被放置在6mm的高度，使得在单个2.8微米磁珠上的力是恒定的（约30-40 PN）31。张力刺激的持续时间是由根据兴趣和其时间尺度活化的分子上的操作者确定。将细胞最后裂解，粘附络合物通过珠粒分离利用磁铁纯化和生物化学分析被处理。这个协议包括的涂覆配体超顺磁珠的制备，和张力通过磁体的应用，随后通过生物化学分析。此外，我们还提供应用于基于整联-ADH数据表明张力的代表性样品esions诱导粘附重塑和改变蛋白质的酪氨酸磷酸化。

<table><tbody><tr><td>Neodymium magnets (on the upper face of 60 mm dish)</td><td>K&amp;amp;J Magnetics, Inc</td><td>DX88-N52</td><td>grade N52 dimension: 1 1/2&quot; dia. x 1/2&quot; thick</td></tr><tr><td>Neodymium magnets (on the lower face of 60 mm dish)</td><td>K&amp;amp;J Magnetics, Inc</td><td>D84PC-BLK</td><td>grade N42 dimension: 1/2&quot; dia. x 1/4&quot; thick Black Plastic Coated&amp;nbsp;</td></tr><tr><td>Dynabeads M280 Tosylactivated</td><td>Thermofisher</td><td>14203</td><td>superparamagnetic beads&amp;nbsp;</td></tr><tr><td>DynaMag-2 Magnet</td><td>Thermofisher</td><td>12321D</td><td></td></tr><tr><td>Fibronectin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F1141-5MG</td><td>Fibronectin from bovine plasma</td></tr><tr><td>Poly-D-Lysine</td><td>Sigma-Aldrich</td><td>P7280-5MG</td><td></td></tr><tr><td>Apo-Transferrin</td><td>Sigma-Aldrich</td><td>T1428-50MG</td><td>Bovine Apo-Transferrin</td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A7906-500G</td><td></td></tr><tr><td>DMEM high glucose, GlutaMAX supplement, pyruvate&amp;nbsp;</td><td>Life Technologies</td><td>31966-021</td><td>DMEM+GlutaMAX-I 500 ml&amp;nbsp;</td></tr><tr><td>60*15 mm culture dish</td><td>Falcon</td><td>353004</td><td></td></tr></tbody></table>

analyzing cell surface adhesion remodeling in response to mechanical tension using magnetic beads

机械敏感细胞表面粘附物使细胞感测与其周围环境的机械性能。最近的研究已经确定了在固定区域既力传感分子和调节谱系特异性基因表达和驱动表型输出力依赖性转录因子。然而，信令网络转换机械张力成生化途径仍然难以捉摸。探索时施加到细胞表面受体的机械张力接合的信号传导途径，可以使用超顺磁性微珠。在这里，我们提出了一个协议利用磁珠力量应用到细胞表面粘附蛋白。使用这种方法，可以通过安装在涂覆配体珠粘附复合物的磁隔离，调查通过各种生物化学方法不仅力依赖性细胞质信号传导途径，而且粘附重塑。这个协议包括配体 - 共聚的制备ated超顺磁珠，和应用程序中定义的拉力接着生物化学分析。此外，我们提供的数据表明应用于基于整合素的粘附张力的代表性样品触发粘附重塑和改变蛋白质的酪氨酸磷酸化。

该技术的原理在图1a中示出。以下的配体缀合，磁珠与细胞孵育20分钟，然后一个永久磁铁用于施加约30-40 PN的拉伸力对各种量的时间。 图1b示出了2.8微米势必MRC5细胞粘附受体的FN包被的磁珠。 细胞裂解后的超顺磁珠洗涤步骤是关键的，并确定纯化的程度。建议三次洗涤最低。 GADPH免疫印迹与长时间曝光可以测试粘附复合物的纯度（图2a）是</stro...

Watch this Scientific Journal Video about Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads at JoVE.com

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

细胞表面粘连是在机械传导的中心，因为它们传输机械张力，并开始参与组织稳态和发展的信号通路。这里，我们提出了解剖被响应于张力激活生化途径，使用包被配体 - 磁珠和施力到粘附受体的协议。

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 ...

analyzing-cell-surface-adhesion-remodeling-response-to-mechanical

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

8.4K 次观看.  Centre de recherche UGA - INSERM U1209 - CNRS UMR. 细胞表面粘连是在机械传导的中心，因为它们传输机械张力，并开始参与组织稳态和发展的信号通路。这里，我们提出了解剖被响应于张力激活生化途径，使用包被配体 - 磁珠和施力到粘附受体的协议。 

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

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis1, atherogenesis2 and wound healing3. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance4-7.
	Single molecule techniques such as optical trapping8, atomic force microscopy9, and magnetic tweezers10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level12. Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.

警队复用单分子蛋白水解测量磁性镊子

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

科研

生物工程

Magnetic Tweezers for the Measurement of Twist and Torque

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques include so-called force spectroscopy approaches such as atomic force microscopy, optical tweezers, flow stretching, and magnetic tweezers. Amongst these approaches, magnetic tweezers have distinguished themselves by their ability to apply torque while maintaining a constant stretching force. Here, it is illustrated how such a &#8220;conventional&#8221; magnetic tweezers experimental configuration can, through a straightforward modification of its field configuration to minimize the magnitude of the transverse field, be adapted to measure the degree of twist in a biological molecule. The resulting configuration is termed the freely-orbiting magnetic tweezers. Additionally, it is shown how further modification of the field configuration can yield a transverse field with a magnitude intermediate between that of the &#8220;conventional&#8221; magnetic tweezers and the freely-orbiting magnetic tweezers, which makes it possible to directly measure the torque stored in a biological molecule. This configuration is termed the magnetic torque tweezers. The accompanying video explains in detail how the conversion of conventional magnetic tweezers into freely-orbiting magnetic tweezers and magnetic torque tweezers can be accomplished, and demonstrates the use of these techniques. These adaptations maintain all the strengths of conventional magnetic tweezers while greatly expanding the versatility of this powerful instrument.

Magnetic tweezers, a powerful single-molecule manipulation technique, can be adapted for the direct measurements of the twist (using a configuration called freely-orbiting magnetic tweezers) and torque (using a configuration termed magnetic torque tweezers) in biological macromolecules. Guidelines for performing such measurements are given, including applications to the study of DNA and associated nucleo-protein filaments.

磁性镊子的扭曲和扭矩的测量

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques ...

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions for many of these proteins are poorly understood. Here we present a simple, rapid method for characterizing the adhesive properties of putative homophilic cell adhesion molecules. Cultured HEK293 cells are transfected with DNA plasmid encoding a secreted, epitope-tagged ectodomain of a cell surface protein. Using functionalized beads specific for the epitope tag, the soluble, secreted fusion protein is captured from the culture medium. The coated beads can then be used directly in bead aggregation assays or in fluorescent bead sorting assays to test for homophilic adhesion. If desired, mutagenesis can then be used to elucidate the specific amino acids or domains required for adhesion. This assay requires only small amounts of expressed protein, does not require the production of stable cell lines, and can be accomplished in 4 days.

Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

珠聚集测定假定的细胞粘附分子的表征

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

新方法本地化记者荧光珠近细胞培养表面的牵引力显微镜

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Magnetic Tweezers in a Microplate Format

Mechanobiology describes how the physical forces and mechanical properties of biological material contribute to physiology and disease. Typically, these approaches are limited single-molecule methods, which restricts their availability. To address this need, a microplate assay was developed that enables mechanical manipulation while performing standard biochemical assays. This is achieved using magnets incorporated into a microplate lid to create multiple magnetic tweezers. In this format, force is exerted across biomolecules connected to paramagnetic beads, equivalent to a typical magnetic tweezer. The study demonstrates the application of this tool with FRET-based assays to monitor protein conformations. However, this approach is widely applicable to different biological systems ranging from measuring enzymatic activity through to the activation of signaling pathways in live cells.

Here, we describe the use of a novel microplate assay to enable mechanical manipulation of biomolecules while performing ensemble biochemical assays. This is achieved using a microplate lid modified with magnets to create multiple static magnetic tweezers across the microplate.

Mechanobiology describes how the physical forces and mechanical properties of biological material contribute to physiology and disease. Typically, these ...

生物化学

High-Speed Magnetic Tweezers for Nanomechanical Measurements on Force-Sensitive Elements

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are therefore poised to be useful in the field of mechanobiology. Since the method commonly relies on image-based tracking of magnetic beads, the speed limit in recording and analyzing images, as well as the thermal fluctuations of the beads, has long hampered its application in observing small and fast structural changes in target molecules. This article describes detailed methods for the construction and operation of a high-resolution MT setup that can resolve nanoscale, millisecond dynamics of biomolecules and their complexes. As application examples, experiments with DNA hairpins and SNARE complexes (membrane-fusion machinery) are demonstrated, focusing on how their transient states and transitions can be detected in the presence of piconewton-scale forces. We expect that high-speed MTs will continue to enable high-precision nanomechanical measurements on molecules that sense, transmit, and generate forces in cells, and thereby deepen our molecular-level understanding of mechanobiology.

Here, we describe a high-speed magnetic tweezer setup that performs nanomechanical measurements on force-sensitive biomolecules at the maximum rate of 1.2 kHz. We introduce its application to DNA hairpins and SNARE complexes as model systems, but it will be also applicable to other molecules involved in mechanobiological events.

高速磁镊，用于力敏元件的纳米力学测量

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are ...

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat soft substrates that deform under cell traction (2D-TFM). TFM relies on the use of linear elastic materials, such as polydimethylsiloxane (PDMS) or polyacrylamide (PA). For 2D-TFM on PA, the difficulty in achieving high throughput results mainly from the large variability of cell shapes and tractions, calling for standardization. We present a protocol to rapidly and efficiently fabricate micropatterned PA hydrogels for 2D-TFM studies. The micropatterns are first created by maskless photolithography using near-UV light where extracellular matrix proteins bind only to the micropatterned regions, while the rest of the surface remains non-adhesive for cells. The micropatterning of extracellular matrix proteins is due to the presence of active aldehyde groups, resulting in adhesive regions of different shapes to accommodate either single cells or groups of cells. For TFM measurements, we use PA hydrogels of different elasticity by varying the amounts of acrylamide and bis-acrylamide and tracking the displacement of embedded fluorescent beads to reconstruct cell traction fields with regularized Fourier Transform Traction Cytometry (FTTC).
To further achieve precise recording of cell forces, we describe the use of a controlled dose of patterned light to release cell tractions in defined regions for single cells or groups of cells. We call this method local UV illumination traction force microscopy (LUVI-TFM). With enzymatic treatment, all cells are detached from the sample simultaneously, whereas with LUVI-TFM traction forces of cells in different regions of the sample can be recorded in sequence. We demonstrate the applicability of this protocol (i) to study cell traction forces as a function of controlled adhesion to the substrate, and (ii) to achieve a greater number of experimental observations from the same sample.

Near-UV lithography and traction force microscopy are combined to measure cellular forces on micropatterned hydrogels. The targeted light-induced release of single cells enables a high number of measurements on the same sample.

使用水凝胶图案化技术控制细胞粘附，用于牵引力显微镜的应用

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat ...

Pattern Generation for Micropattern Traction Microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

微图案牵引显微镜的图案生成

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...

Single Cell Durotaxis Assay for Assessing Mechanical Control of Cellular Movement and Related Signaling Events

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate underlying a cell must be manipulated. While hydrogels with graded stiffness and long-term migration assays have proven useful in durotaxis studies, immediate, acute responses to local changes in substrate tension allow focused study of individual cell movements and subcellular signaling events. To repeatably test the ability of cells to sense and respond to the underlying substrate stiffness, a modified method for application of acute gradients of increased tension to individual cells cultured on deformable hydrogels is used which allows for real time manipulation of the strength and direction of stiffness gradients imparted upon cells in question. Additionally, by fine tuning the details and parameters of the assay, such as the shape and dimensions of the micropipette or the relative position, placement, and direction of the applied gradient, the assay can be optimized for the study of any mechanically sensitive cell type and system. These parameters can be altered to reliably change the applied stimulus and expand the functionality and versatility of the assay. This method allows examination of both long term durotactic movement as well as more immediate changes in cellular signaling and morphological dynamics in response to changing stiffness.

Mechanical forces are important for controlling cell migration. This protocol demonstrates the use of elastic hydrogels that can be deformed using a glass micropipette and a micromanipulator to stimulate cells with a local stiffness gradient to elicit changes in cell structure and migration.

用于评估细胞运动和相关信号事件的机械控制的单细胞Durotaxis测定

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate ...

生物学

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.