Name: single cell durotaxis assay for assessing mechanical control of cellular movement and related signaling events
Uploaded: 2019-08-27T14:45:00
Description: Watch this Scientific Journal Video about Single Cell Durotaxis Assay for Assessing Mechanical Control of Cellular Movement and Related Signaling Events at JoVE.com

1. 用嵌入式荧光微球制造可变形的聚丙烯酰胺水凝胶
注：方向描述直径为 22 μm、厚度约为 66 μm 的 25 kPa 水凝胶的聚合。可以修改每个或所有这些参数，并在表 1和备注17中找到这样做的方向。
<ol><li>激活玻璃底餐具或盖玻片
	<ol>
<li>准备结合硅烷工作溶液，用于激活玻璃底成像盘或适合活细胞成像室的盖玻片。混合 950...

<ol>
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这里演示的是一个可重复的单细胞durotaxis测定，允许评估细胞在响应急性机械提示时改变其迁移行为的能力。该技术还可用于荧光显微镜和适当的融合蛋白或生物传感器，以检查亚细胞信号和细胞骨骼事件在机械刺激的几秒钟内或超过较长的时间刻度杜罗法运动。了解细胞与其环境的关系涉及研究该环境的化学和机械方面的影响。虽然可能难以掌握，但这种杜罗塔斯测定可以广泛用于理解细胞对?...

在过去的几十年里，细胞环境的机械特性的重要性在细胞生物学中得到了越来越多的认可。不同的组织和细胞外基质具有不同的相对刚度，当细胞在全身迁移时，它们会导航这些变化，使用这些机械特性来引导它们1，2，3,4,5,6,7.细胞使用给定组织的刚度在发育、伤口愈合和癌症转移等过程中告知其活动行为。然而，允许感觉和响应这些机械输入的分子机制在很大程度上仍然未知1，2，3，4，5，6,7.
为了研究细胞对物理环境线索的反应机制，必须操纵基底基体附着细胞的刚度或刚度。2000年，罗春敏、王玉丽及其同事研制出一种测定8，通过拉伸可变形的细胞外基质（ECM）涂层聚丙烯酰胺，直接测试单个细胞对机械线索变化的移动反应。水凝胶，细胞被镀。细胞表现出对向更硬基质迁移的偏好，他们称之为"杜罗塔克"。
自2000年提交原始报告以来，还采用了许多其他技术来研究杜罗塔。通过将凝胶铸造在硬质特征（如聚苯乙烯珠9或硬聚合物柱10）上，或通过在玻璃盖玻片11的边缘聚合基板来制造陡峭的刚度梯度，从而形成机械性'步进边界'。或者，采用较浅但固定刚度梯度的水凝胶由多种方法制成，例如微流体装置12、13或并排水凝胶溶液液滴创建的交联物梯度不同的刚度 8 ，或水凝胶与光活联处理与分级紫外线光曝光，以创建线性刚度梯度 14 ， 15 。这些技术已经被用来研究随着时间的推移，大量的二维细胞运动。然而，这些特征通常是在细胞电镀之前制造的，在实验过程中，它们的性能保持一致，依靠随机细胞运动对机械梯度进行采样。这些技术都无法观察细胞行为在急性机械刺激下的快速变化。
为了观察细胞对机械环境急剧变化的反应，单细胞durotaxis测定具有几个优点。在这些测定中，通过用玻璃微移液器将底层基板从细胞中拉出，从而引入细胞基质张力的方向梯度，从而给单个细胞一种急性的机械刺激。然后，活细胞相对比显微镜观察移动行为的变化，如迁移的速度或方向。这种方法有助于直接观察机械刺激和细胞迁移之间的因果关系，因为它允许快速、反复地操作张力梯度的方向和幅度，并评估其细胞实时反应。此外，该方法还可用于机械刺激细胞表达荧光融合蛋白或生物传感器，以可视化被怀疑参与机械感应和杜罗塔斯。
这项技术已被研究杜罗塔克8，16的小组使用，并在这里描述，因为它已被豪实验室改造，以研究SKOV-3卵巢癌细胞的杜罗法行为和分子机制，下杜罗塔17。此外，还描述了一种改进方法，用于在细胞培养表面附近用单层甚至荧光微球制造水凝胶;这有利于微移液器生成的应变梯度的可视化和优化，并可能允许通过牵引力显微镜评估细胞收缩性。

<table>
	<tbody>
		<tr>
			<td>Acrylamide 40 %&#160;</td>
			<td>National Diagnostic</td>
			<td>EC-810</td>
			<td></td>
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			<td>Ammonium Persulfate&#160;</td>
			<td>Fisher</td>
			<td>BP179-25</td>
			<td></td>
		</tr>
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			<td>BD20A High frequency generator</td>
			<td>Electro Technic Products</td>
			<td>12011A</td>
			<td>115 V -&#160;Handheld Corona Wand</td>
		</tr>
		<tr>
			<td>Bind Silane (y-methacryloxypropyltrimethoxysilane) (</td>
			<td>Sigma Aldrich</td>
			<td>M6514</td>
			<td></td>
		</tr>
		<tr>
			<td>Bis-acrylamide 2%&#160;</td>
			<td>National Diagnostic</td>
			<td>EC-820</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries</td>
			<td>World Precision Instruments</td>
			<td>1B100-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Branson 2510 Ultrasonic Cleaner</td>
			<td>Bransonic</td>
			<td></td>
			<td>40 kHz frequency</td>
		</tr>
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			<td>Coarse Manipulator</td>
			<td>Narshige</td>
			<td>MC35A</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM without phenol red</td>
			<td>Sigma Aldrich</td>
			<td>D5030</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual-Stage Glass Micropipette Puller</td>
			<td>Narshige</td>
			<td>PC-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Pharmco-aaper</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (Qualified One Shot)</td>
			<td>Gibco</td>
			<td>A31606-02</td>
			<td></td>
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		<tr>
			<td>Fibronectin&#160;</td>
			<td>EMD Millipore</td>
			<td>FC010</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluospheres Carboxylate 0.2 um&#160;</td>
			<td>Invitrogen</td>
			<td>F8810, F8807, F8811</td>
			<td></td>
		</tr>
		<tr>
			<td>Fugene 6</td>
			<td>Roche</td>
			<td>1815091</td>
			<td>1.5 ug DNA / 6uL fugene 6 per 35mm dish</td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid</td>
			<td>Fisher Chemical</td>
			<td>A38SI-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bottom Dish</td>
			<td>CellVis</td>
			<td>D60-60-1.5-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Coverslip</td>
			<td>Electron Microscopy Sciences</td>
			<td>72224-01</td>
			<td>22 mm, #1.5</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>JT Baker</td>
			<td>9535-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Hellmanex III&#160;Special cleaning concentrate</td>
			<td>Sigma Aldrich</td>
			<td>Z805939</td>
			<td>Used at 2% in ddH2O for cleaning coverslips</td>
		</tr>
		<tr>
			<td>HEPES powder</td>
			<td>Sigma Aldrich</td>
			<td>H3375</td>
			<td>Make 50mM HEPES buffer, pH 8.5</td>
		</tr>
		<tr>
			<td>Intelli-Ray 400 Shuttered UV Flood Light</td>
			<td>Uviton International</td>
			<td>UV0338</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher Chemical</td>
			<td>A417-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Microforge</td>
			<td>Narshige</td>
			<td>MF900</td>
			<td></td>
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			<td>Micromanipulator</td>
			<td>Narshige</td>
			<td>MHW3</td>
			<td></td>
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		<tr>
			<td>Mineral Oil</td>
			<td>Sigma Aldrich</td>
			<td>M5904</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanopure Life Science UV/UF System</td>
			<td>Barnstead</td>
			<td>D11931</td>
			<td>ddH2O</td>
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		<tr>
			<td>Nikon Eclipse Ti</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OptiMEM</td>
			<td>Invitrogen</td>
			<td>31985062</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Bemis Company, Inc</td>
			<td>PM-992</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td></td>
			<td></td>
			<td>139 mM NaCl, 2.5 mM KCl, 28.6 mM Na2HPO4, 1.6 mM KH2PO4, pH 7.4</td>
		</tr>
		<tr>
			<td>Platelet Derived Growth Factor-BB (PDGF-BB)</td>
			<td>Sigma Aldrich</td>
			<td>P4056</td>
			<td></td>
		</tr>
		<tr>
			<td>Ref52</td>
			<td></td>
			<td></td>
			<td>Rat embryonic fibroblast cell line; Culture in DMEM + 10% FBS</td>
		</tr>
		<tr>
			<td>Ringer&#39;s Buffer</td>
			<td></td>
			<td></td>
			<td>134 mM NaCl, 5.4 mM KCl, 1 mM MgSO4, 2.4 mM CaCl2, 20 mM HEPES, 5 mM D-Glucose, pH 7.4</td>
		</tr>
		<tr>
			<td>SKOV-3</td>
			<td>American Type Culture Collection</td>
			<td></td>
			<td>Culture in DMEM + 10% FBS</td>
		</tr>
		<tr>
			<td>Sulfo-SANPAH&#160;</td>
			<td>Covachem</td>
			<td>&#160;12414-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tabletop Plasma Cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-32G</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED&#160;</td>
			<td>Sigma Aldrich</td>
			<td>T9281-50</td>
			<td></td>
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single cell durotaxis assay for assessing mechanical control of cellular movement and related signaling events

杜罗拉茨是细胞感知和响应紧张梯度的过程。为了在体外研究这个过程，必须操纵细胞基础基板的刚度。虽然具有分级刚度和长期迁移检测的水凝胶已证明在durotaxis研究中有用，但是对基质张力局部变化的即时急性反应允许对单个细胞运动和亚细胞信号事件进行重点研究。为了重复测试细胞对底层基底刚度的感知和反应能力，采用了一种改进的方法，用于对在可变形水凝胶上培养的单个细胞应用增加张力的急性梯度，从而实现实时操作刚度梯度的强度和方向传授给有问题的细胞。此外，通过微调测定的细节和参数，如微移液器的形状和尺寸或应用梯度的相对位置、位置和方向，可以优化测定，以进行机械研究。敏感细胞类型和系统。这些参数可以改变，以可靠地改变应用的刺激，并扩展测定的功能和多功能性。这种方法允许检查长期的柔化运动，以及细胞信号和形态动力学的更直接的变化，以响应不断变化的刚度。

通过制备微移液器（图1）和如上所述的拉力生成（图2和图3）标准化，为多个细胞系确定了最佳的二维战术条件。使用这种技术，如图4所述，SKOV-3卵巢癌细胞17和Ref52大鼠胚胎纤维细胞（图5）都朝着玻璃微移液器应用的梯度增加的刚度方向发展。除了杜罗塔，这种方?...

Watch this Scientific Journal Video about Single Cell Durotaxis Assay for Assessing Mechanical Control of Cellular Movement and Related Signaling Events at JoVE.com

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

机械力对于控制细胞迁移非常重要。该协议演示了弹性水凝胶的使用，可以使用玻璃微移液器和微操纵器变形，以刺激具有局部刚度梯度的细胞，从而引起细胞结构和迁移的变化。

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 ...

人们越来越认识到机械微环境对细胞信号和迁移的影响。需要使用独特的实验方法向单个细胞提供机械刺激。该技术允许实时动态操作细胞下方的机械微环境。允许观察细胞迁移行为和亚细胞信号事件的机械调节变化。与我展示这个程序的将是尼拉·奈姆，一个博士后和约翰森·帕特森，一个来自我们实验室的技术人员。首先使用 Chorono-1 激活每个底盖滑的玻璃表面 20 秒，然后立即将 50 微升的绑定硅烷工作溶液覆盖到每个盖滑上。让溶液干燥10分钟，然后用95%乙醇冲洗盖滑两次，用异丙醇冲洗两次。然后，让盖滑滑风干燥约 20 分钟。对于荧光微珠沉积，首先对工作微珠溶液进行一小时的声波化。在声波化结束前 15 分钟，将激活的盖板垂直放在陶瓷盖滑架中，然后将支架转移到桌面等离子清洁剂中，进行室内空气等离子处理三分钟。接下来，将石蜡薄膜片片放在60毫升 Petri 盘盖中，将盖滑到薄膜上，轻轻敲击每个盖玻片，以确保薄膜与盖玻片之间的良好接触。然后，将工作珠溶液的 150 微升添加到每个盖玻片的顶部，并立即从盖玻片的一侧吸气乙醇溶液，将珠子留在盖玻片上。要在水凝胶制备后立即铸造水凝胶，请将 25 微升水凝胶液滴添加到每个底部盖玻片的激活侧，并立即将珠片顶部盖玻片、珠侧向下翻转到液滴上。让凝胶聚合 30 分钟，然后使用钳子轻轻取下盖玻片。然后，每次洗涤时用新鲜 50 miliMoler Hepes 冲洗 3、 5 分钟洗涤凝胶。为了激活水凝胶表面，在新鲜的 50 miliMoler Hepes 中孵育凝胶，辅以 0.4 miliMolar Sulfo Sanpah，并立即将凝胶暴露在封闭区域的紫外线拱灯中 90 秒。在激活结束时，用新鲜 Hepes 洗涤凝胶三、五分钟，并在 37 摄氏度下用每毫升纤维素 20 微克处理激活的水凝胶，一小时。在孵育结束时，吸进多余的纤维素溶液，并在PBS中清洗三次凝胶，每次洗涤五分钟。最后一次洗涤后，将水凝胶放在少量 PBS 中，并在组织培养罩的紫外线下对凝胶进行 15 分钟的消毒。然后，在新鲜的PBS中清洗凝胶一次。对于细胞电镀，种子2.1倍10的四个感兴趣的细胞在三毫升的中等每水凝胶，并允许细胞在37摄氏度粘附4至18小时。当细胞进行平衡时，使用微移液器拉拔器将直径一毫米的1毫米长100毫米的硅酸盐玻璃微移液器拉拉，获得超过两毫米的锥度，在第一毫米内直径减至约50毫米，并在最后一毫米内延伸至长、平行的10毫米直径管。接下来，将移液器加载到微叉中，并塑造移液器，使移液器具有 15 微米的钝化尖端，该尖端封闭在 250 微米部分的末尾，与移液器的其余部分弯曲约 35 度天使。弯道处的近似直径应约为 30 微米，以赋予尖端强度。接下来，将水凝胶放在倒置显微镜的舞台上。用矿物油覆盖介质，并选择 10 倍目标。用70%酒精对准备好的微烟斗进行消毒。然后，插入微移液器支架，将挂钩指向盘子，并使用过程操纵器降低移液器，直到挂钩刚刚接触液体表面。将显微镜聚焦在粘在凝胶顶部的细胞上，注意目标没有撞击样品或阶段的危险，将焦点放在凝胶上方，以找到微移液器的尖端。旋转移液器，直到尖端垂直于焦点平面，使微移液器的钝尖端向下倾斜，并必要时重新聚焦移液器的尖端。将焦点对回到细胞层，以测量移液器离凝胶表面有多远，并聚焦回凝胶和移液器尖端之间的平面。然后，缓慢降低移液器以到达中间焦平面，并将放大倍率提高至实验中使用的放大倍率，然后降低移液器，直到它悬停在水凝胶表面上方。对于微机械手校准，成像一个没有细胞的区域，一个区域与珠子，但没有操作，与移液器接合凝胶，并与接合移液器拉凝胶。然后，使用图像 J 将无珠场与没有啮合的珠场进行比较，将带凝胶的珠场与拉凝胶进行比较，并计算对凝胶施加的相对珠子位移和力。要进行杜罗塔西测定，监测一组细胞30分钟，以识别以定向方式移动的细胞。选择一个向一个方向稳步移动的单元格，并监控所需帧速率的单元格，再持续 30 分钟。接下来，将移液器定位在选定细胞前缘近侧约 50 微米，并移动微操纵器，使凝胶正交变形到细胞的行驶方向。然后，观察细胞随着时间的推移，因为它对水凝胶刚度的急性局部梯度作出反应。使用这种技术，如证明，大鼠胚胎成纤维细胞走向增加硬度的梯度应用玻璃微移液器。大鼠胚胎成纤维细胞，在125千帕氏多丙烯酰胺凝胶上短暂表达文库林张力传感器，也证明在40分钟内在拉伸方向形成焦点粘附。福斯特共振能量转移分析显示，当呈现急性非病刺激时，局部到焦点粘附的葡萄子素会立即改变张力。扩大这种测定的效用，以观察亚细胞信号事件，以回应杜洛特刺激。如果您多次尝试拉伸细胞，或者微需要在检测过程中滑落，请从新细胞开始，以避免传递多种可变的机械刺激。除了用于检测杜罗塔西的用途外，该技术还可以与基因方法（如生物传感器、RNAI 或基因编辑）相结合，以帮助描述机械转导所涉及的分子机制。

Research

JoVE Journal

Biology

Cellular Toxicity of Nanogenomedicine in MCF-7 Cell Line: MTT assay

Nanogenomedicine在MCF - 7细胞的细胞毒性：MTT法

Cytotoxicity of the futuristic nanogenomedicine (e.g., short interfering RNA and antisense) may hamper its clinical development. Of these, the gene-based ...

科研

生物学

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

成像摹蛋白偶联受体（GPCR）介导的信号，控制趋化粘菌</em

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

FRET显微镜在活细胞单分子生物传感器的信号事件的实时监测

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

为外植体含量的颅间充质细胞行为评定

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

机械刺激引起的钙波传播的细胞单层：牛角膜内皮细胞的例子

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Single-cell Microinjection for Cell Communication Analysis

Cell的传播分析单细胞显微注射

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

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 ...

Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast

在裂殖酵母中Cytokinetic活动的时空分析

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and ...

Single-cell Microfluidic Analysis of Bacillus subtilis

Single-cell Microfluidic Analysis of Bacillus subtilis

单细胞微流控芯片分析枯草芽孢杆菌

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers ...