Name: simple polyacrylamide-based multiwell stiffness assay for the study of stiffness-dependent cell responses
Uploaded: 2015-03-25T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses at JoVE.com

This work was funded by start-up funds provided to Dr. Silviya Zustiak by Saint Louis University as well as by a President&#8217;s Research Fund (PRF) grant awarded to Dr. Silviya Zustiak by Saint Louis University. We thank Naveed Ahmed and Keval Shah for technical assistance.

1.准备水凝胶有关的解决方案和等分<ol><li>制备的聚丙烯酰胺凝胶前体溶液。 <ol><li>通过混合丙烯酰胺（A）中制备的聚丙烯酰胺凝胶前体溶液（40％w / v的，M R71.08克/摩尔），交联剂双丙烯酰胺（B）（2％w / v的，M R154.17克/摩尔），和DE-离子水在表1中规定的体积百分比。 注意:这些解决方案能在大批量制备，并储存在4℃下进行长达数月。 <ol><li>注意:丙烯酰胺是在吸入或摄入有毒，特别是，当以粉末形式:因此，优选使用40％w / v的溶液，以降低毒性风险。只处理而穿着防护服，如手套，护目镜，以及白大褂。在储存在冰箱的耐光，密闭的容器中（&lt;23°C，通风良好的地方）。 </li><li>注意:双 - 丙烯酰胺是在吸入或摄入有毒，特别是，粉末形式时:因此，优选使用2％w / v的溶液，以降低毒性风险。只处理而穿着防护服，如手套，护目镜，以及白大褂。在储存在冰箱（&lt;4°C，通风良好的地方）光性，密闭容器内。 </li></ol></li></ol></li><li>制备和 N -Sulfosuccinimidyl -6-（4&#39;-叠氮基-2&#39;-硝基苯基氨基）己酸的使用量（磺基SANPAH，M R492.40克/ mol）的等分试样。注意:磺基SANPAH造成严重眼刺激;手套和适当的眼部或面部防护处理。储存于-20℃，在​​收到和前分装。 <ol><li>以存储磺基SANPAH:50毫克/毫升溶解磺基SANPAH在dimethylsulfoxane（DMSO）中。分装原液到每管20微升50微离心管中。在干冰上，或在液氮中闪冻（任选的但优选的步骤，以保持最大效率交联剂），并存储在-80℃。 注:CA等分N为存放数个月。 </li><li>使用磺基SANPAH:解冻的等分试样简要和稀释在480微升去离子水。立即使用。磺SANPAH迅速水解水:所以采取谨慎快速执行上述所有步骤。 </li></ol></li><li>准备过硫酸铵（Mr为228.18克/摩尔）等分。 注意:过硫酸铵引起眼睛，皮肤和呼吸系统有刺激性。处理时，佩戴合适的个人防护装备。处理过硫酸铵粉末在化学通风橱中。粉储存在干燥，通风良好的地方。一旦分装，稀溶液可用于在工作台上。 <ol><li>溶解过硫酸铵在去离子水以达到最终的过硫酸铵浓度为10％w / v的。分装和储存在-20℃。解冻使用前立即。 </li></ol></li><li>制备胶原蛋白I型的解决方案。 <ol><li>制备0.2mg / ml的胶原溶液稀释储备所以lution在pH为7.4的1×磷酸盐缓冲盐水（PBS）中。储存在冰短暂或稀释后立即使用。 </li></ol></li></ol> 2.水凝胶的制备（参见图1） <ol><li>制备的疏水载玻片上。 <ol><li>放置几滴疏水溶液在玻璃板上，并用纸巾在表面上传播。让空气干燥，并再次用纸巾擦拭纸拉平疏水涂层。 注:易燃内阁在室温储存疏水性的解决方案。处理时佩戴个人防护装备。在通风良好的地方工作。 </li></ol></li><li>制备的柔性塑料支撑。 <ol><li>切割柔性塑料支撑以匹配疏水涂覆玻璃板的尺寸。 </li><li>通过轻轻搔抓用尖锐的工具的表面上，如手术刀马克挠性塑料支撑的疏水侧。 注意:一旦凝胶 - 这是完全透明的 - 烘的划痕将有助于区分哪些灵活的塑料支撑侧含有凝胶。 注:柔性塑料支撑具有疏水和亲水侧。一旦沉积，聚丙烯酰胺永久坚持的塑料支撑这有利于方便后续处理水凝胶的亲水性的一面。 </li></ol></li><li>凝胶法制备（例如卷，给出了5毫升凝胶前体解决方案）。 注:5毫升将足以产生〜40的凝胶时的凝胶的初始厚度为0.5毫米。更多的凝胶可以生产如果凝胶厚度减小。 <ol><li>放置4972.5微升期望的最终浓度（ 表1）的聚丙烯酰胺的前体溶液到50毫升锥形管中。放置在脱气室30分钟以打开盖子。 注:氧气用作自由基捕集和当存在时，其抑制聚合。 </li><li>添加25微升10％w / v的过硫酸铵（参照点1.3）以达到最终的0.05％过硫酸铵浓度。 </li><li>加入2.5微升的N，N，N&#39;，N&#39;-四甲基乙二胺（TEMED，M R116.24克/摩尔）于脱气的凝胶溶液以达到0.5％的最终TEMED浓度。 注:储存TEMED在易燃内阁在室温。穿着适当的个人防护设备时，处理化学通风柜。保持在惰性气体密闭的，因为它是高度空气和水分敏感。 </li><li> 5倍 - 吹打向上和向下轻轻3混合的解决方案。不要旋涡振荡，以避免氧扩散到凝胶前体溶液。 </li><li>吸管将凝胶溶液涂布在柔性塑料支撑和三明治用疏水涂层玻璃载片的亲水侧。具有所需厚度（ 例如，0.5mm）的硅隔板分开的两个幻灯片。离开的聚合物前体溶液少量（约100微升）在50毫升锥形管作为凝胶化的指标来使用。 注意:任何间隔可被使用。例如，封口膜的单个条给出了100的最终的溶胀凝胶的厚度 - 120微米。 </li><li>将另一玻璃板上盖的弹性塑料支撑/凝胶三明治聚合后确定的，即使水凝胶表面。 </li><li>让凝胶聚合​​45分钟，尽管更少的时间，可用于较高的％（重量）的凝胶，如果需要的。以确定该凝胶已聚合，观察在50ml锥形管中的剩余溶液;如果剩余的溶液已凝胶化，则很可能该凝胶在玻璃板已被启动以及。但是请注意，模具过早开放将防止完全聚合。 </li><li>一旦凝胶形成，剥离的柔性塑料支撑在顶部具有共价附着的聚丙烯酰胺凝胶，并设置凝胶朝上风干。 注:对流或热干燥，也可用于加速此步骤。一旦干燥到柔性塑料支撑，该凝胶可以是存储无限期ð。 <ol><li>纪念弹性塑料的支持，其中聚丙烯酰胺凝胶并没有因为泡沫形成的裸露的斑点。标记而凝胶仍水合，因为这将融合在一起的柔性塑料支撑一旦凝胶干燥。 </li></ol></li></ol></li></ol> 3.多孔板组装，胶原蛋白涂层和灭菌<ol><li>多孔板组件。 <ol><li>一旦干燥，切成PA凝胶成所需的形状。 <ol><li>对于一个96孔板中，使用重型打孔器，直径为〜6mm之间。使用重型切纸机切断的凝胶成正方形或矩形形状。或者，用剪刀。 </li></ol></li><li>根据制造商的说明每96孔板制备约500微升的PDMS。胶水凝胶到多孔板的底部，放置一个小墨滴（〜5微升）聚二甲基硅氧烷（PDMS）中的的中央各孔中。使用镊子，将一个聚丙烯酰胺ë凝胶在各孔中，柔性塑料支撑面朝下。为了让PDMS治愈，留下的组装板在37℃最少4小时的。 </li></ol></li><li>聚丙烯酰胺凝胶的胶原蛋白涂层。 <ol><li>用移液管，将少量 - 磺基 - SANPAH（7 8微升）（参照点1.2.2）的各孔中，涡流从一侧到另一侧涂覆凝胶表面均匀。工作迅速自磺基SANPAH并不稳定在水中。 </li><li>放置孔板下高强度UV灯（强度= 37毫瓦，λ= 302 - 365 nm）的5分钟。冲洗用PBS凝胶以除去过量的磺基 - SANPAH。 </li><li>吸管将50μl0.2mg / ml的I型胶原溶液（参照点1.4）到每个孔中。留在4℃下覆盖在RT至少2小时或O / N的板。 注意:为了加快胶原涂层的过程中，移液管可用于添加胶原溶液。通常情况下，1 - 2液滴的溶液应该是足以完成LY覆盖凝胶表面。 </li><li>离开孔板在RT至少2小时，以允许胶原粘接。 </li><li>用PBS冲洗以除去过量的胶原蛋白溶液和UV（λ= 200纳米）下消毒的组织培养罩2小时。 </li><li>泡在完全培养基（参见4.1节的培养基成分）O / N来滋润和平衡的凝胶。用于小区在冰箱立即或商店播种长达2天。 </li></ol></li></ol>在PA刚度试验4.细胞种植注意:虽然典型的常见的哺乳动物细胞系，在这个部分中所概述的协议进行具体的乳腺癌MDA-MB-231细胞系中使用（ 见图4和5）。 <ol><li>通过暴露收集从组织培养烧瓶中的细胞，以5％的胰蛋白酶/ EDTA进行5分钟，在37℃。使用〜80微升胰蛋白酶/ EDTA的每培养瓶中区的每厘米2;例如，使用2个ml胰蛋白酶/ EDTA的FORA T25细胞培养瓶中。重悬浮收集的细胞中，在所期望的最终细胞浓度补充有10％胎牛血清（FBS）和1％青霉素/链霉素的完全培养基。考虑到细胞倍增时间，以及时间的细胞将被接种上测定的长度，选择适当的子汇合细胞浓度。确保补充介质是足以完全浸没水凝胶。 注意:由于该水凝胶已预平衡的培养基（参见步骤3.2.6）加入100μl体积应该是足够的。用于多孔板的典型介质体积应足以自凝胶已预平衡的培养基在上一步。 注:该测定是适当的任何附着依赖性细胞类型。 <ol><li>通过使用血细胞计数器计数倒置显微镜下的细胞数。负载10微升细胞悬液到每个血球端口和至少8个象限​​平均细胞计数。为了得到最终的细胞浓度，以10 4乘以细胞计数。 注:当在每个血球细胞象限数为20的最佳细胞计数结果获得 - 50。 </li></ol></li><li>培养在37℃下将细胞到PA的刚度测定的加湿培养箱和5％的CO 2。更换介质每2 - 3天。 5分钟需要通过暴露于5％胰蛋白酶/ EDTA，在37℃时收集细胞。使用〜80微升胰蛋白酶/ EDTA的每组织培养瓶的厘米2。在所有电池操作的步骤，特别注意不要破坏凝胶面:吸或吸液管中通过略微倾斜的多孔板侧身触摸枪头到侧壁各井，而不是触摸水凝胶。 注:除采取特别小心不要损坏在标准的组织培养处理的水凝胶表面，将细胞接种到刚性测定法可以被操纵以同样的方式，就好像它们接种在到正规的多孔板。 </li></ol> 5.成像细胞接种于PA刚度分析<ol><li>图像细胞直接作用于PA刚度检测。 注意:任何显微镜 - 倒置，荧光，或共焦，可用于细胞成像。 注意:用于构造刚度试验的软塑料的支持是完全透明的，不autofluoresce或干扰细胞成像。然而，即使在柔性塑料支撑本身是透明的，成像能力将通过物镜的工作距离的限制。柔性塑料支撑体的厚度为0.23毫米，10X物镜的一个典型的工作距离是〜4毫米，大幅降低为较高倍率。 <ol><li>活细胞成像，在显微镜板夹和图像位置PA刚度检测。保持成像会议下2小时，或使用配有环境室长成像倍的显微镜。 </li><li>当活细胞IM老化是不是目的，固定的细胞在4％的甲醛溶液补充有0.1％的清洁剂。浸泡细胞固定液在室温下最少2小时的。用PBS冲洗两次。丢弃固定液浪费在指定的废物容器。 注:细胞可以立即被成像或存储浸没在PBS中，在4℃下进行最多2周。注意:甲醛是对吸入和接触有毒。处理与化学通风柜手套。 注:细胞固定协议具有基于所述随后的细胞染色和处理协议被选择，因为某些固定剂损坏某些细胞蛋白。 </li><li>对于荧光成像，染色固定的细胞具有所需的细胞在多孔板直接染色。图像立即。 </li></ol></li></ol>

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The authors have nothing to disclose.

聚丙烯酰胺凝胶，最初开发用于电泳28现在常规用作细胞培养基材来研究衬底刚度对细胞形态，运动性，并且除其他细胞特征的通信3,24,29的影响。聚丙烯酰胺允许操纵衬底刚度以包含在身体的所有软组织的刚度（0.3 - 300千帕）1，在聚合物前体浓度的简单改变（ 图2，表1中 ，也见参考文献17,25,26）。耦合到一个事实，即PA的凝胶是相当便宜和简单的制备...

大多数组织在体内是软的粘弹性材料具有的杨氏模量为0.1千帕为脑至100千帕软软骨，然而，大多数体外细胞研究的是组织培养聚苯乙烯（TCP）的进行，其具有〜1京帕的模量1，本刚度匹配极大地影响方式细胞所处的环境做出反应。越来越多的研究机构是这样的专用于阐明对各种细胞类型，2,3-包括干细胞。4，结果的命运衬底刚度的效果，多水凝胶已经开发了在刚性依赖的细胞的帮助理解生物学包括聚丙烯酰胺（PA），5-7聚乙二醇（PEG），8,9-二甲基硅氧烷（PDMS），10和藻酸盐。11虽然这种衬底刚度对细胞命运产生重大影响的证据正在增长，大多数研究的进行上小规模用少量第amples。在基板刚度的细胞类型或环境条件是罕见的数组效果系统，多维的研究。12 几个有前途的高通量凝胶技术已被开发出来，其中包括基于PEG的微阵列，用于生产琼脂糖凝胶微珠，14或微和纳米棒，其中刚度由微米棒的直径和高度调制13的微流体装置15不过的技术来制备这样的基体是复杂的，并提供给限于实验室的数目。许多研究涉及刚度调制细胞应答使用聚丙烯酰胺（PA）的凝胶，这是不仅便宜和简单的实现，但也显示出杨氏模量，即0.3的生理学相关范围- 300千帕16-22但是，现有的方法来制造的PA凝胶为细胞培养是劳动密集型的，因此制备ARED小批量。从该凝胶中必须准备的规定部分与P​​A的凝胶作为细胞底物的制备相关的困难茎:1）在不存在氧，以允许完全聚合，2）具有平坦和光滑的表面，以允许均匀的细胞附着和铺展，和3）永久地固定在细胞培养皿为了防止浮动的底部。 几个小组已尝试产生的PA的凝胶为细胞培养在大批量。塞姆勒等人制备的厚片的PA凝胶中，将其"切断"用打孔器并置于96孔板中。23然而，该方法仅限于更硬的凝胶， 即 &gt; 1千帕在杨氏模量，因为较软的凝胶是"粘性"，难以切割，且容易损坏。 MIH 等人开发出了更复杂的技术，它允许对凝胶直接聚合在玻璃底多孔板。 <s达&gt; 6这是通过浇注胶解决方案集成到功能化玻璃底板和"夹"他们自定义的玻璃罩阵列形成凝胶达到6虽然非常有前途的，轻微的边缘效应仍在使用这种技术观察。此外，该技术需要定制设计的阵列不立即向许多实验室以及昂贵玻璃底多孔板访问。 本文介绍了一种简单而廉价的方式来组装PA凝胶中，可以很容易地通过任何实验室多孔板。这里，一个挠性塑料支撑被利用，其中有两个侧边 - 一个疏水性的，这是斥PA凝胶，和亲水性的，在沉积该共价结合的PA的凝胶。一旦PA凝胶片被淀积并永久地固定到柔性塑料支撑，它使得能够处理任何厚度或硬度的凝胶和切削成任何所需的形状。这APPRoach不仅产生定制的塑料"盖玻片&#39;的尺寸不否则市售，也省却了必要的预治疗玻璃表面，无论是玻璃盖玻片或昂贵的玻璃底多孔板的孔中，具有PA的粘合溶液，这是一个繁琐和耗时的工序。最后，均匀的PA的凝胶片材可以在大批量制备并存储的去水合数月。 总之，这里介绍的测定法是一种改进的几个方面的现有的方法。首先，多孔板组装的过程中是有效的，并且所需要的材料的总成本是低的。其次，水凝胶生产大批量在一个单一的均匀的凝胶膜。最后，仅是商业上可用的材料是必需的。该测定的效用是通过探索基板刚度对细胞形态的影响，传播区域所示。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagents&lt;/strong&gt;</td></tr><tr><td>40% Acrylamide</td><td>Bio-Rad</td><td>161-0140</td><td></td></tr><tr><td>2% Bis-acrylamide</td><td>Bio-Rad</td><td>161-0142</td><td></td></tr><tr><td>Ammonium Persulfate</td><td>Bio-Rad</td><td>161-07000</td><td></td></tr><tr><td>TEMED</td><td>Sigma Aldrich</td><td>T9281</td><td></td></tr><tr><td>Sulfo-SANPAH</td><td>Thermo Scientific</td><td>22589</td><td></td></tr><tr><td>Collagen Type 1, from Rat tail, 3.68 mg/ml</td><td>BD Biosciences</td><td>354236</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Fisher Scientific</td><td>BP231-100</td><td></td></tr><tr><td>Hydrophobic solution &amp;#8212; Repel Silane</td><td>GE Healthcare Bio-Sciences</td><td>17-1332-01</td><td></td></tr><tr><td>PBS (1x), pH 7.4</td><td>HyClone</td><td>SH30256.01</td><td></td></tr><tr><td>Polydimehylsiloxane (PDMS) [Slygard 182 Elastomer Kit]</td><td>Elsworth Adhesives</td><td>3097358-1004</td><td></td></tr><tr><td>Tyrpsin/EDTA (10x)</td><td>Sigma Aldrich</td><td>44174</td><td></td></tr><tr><td>RPMI-1640 Medium (1x)</td><td>HyClone</td><td>SH30027-02</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>HyClone</td><td>SH30073-03</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>MP Biomedicals</td><td>1670046</td><td></td></tr><tr><td>Detergent: Triton-X</td><td>Sigma Aldrich</td><td>T8787</td><td></td></tr><tr><td>Formaldehyde 37% Solution</td><td>Sigma Aldrich</td><td>F1635</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma Aldrich</td><td>A2153</td><td></td></tr><tr><td>BSA-based cell adhesion blocking kit &amp;#8212; ECM Cell Adhesion Array Kit</td><td>Chemicon International</td><td>ECM540</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Disposable lab equipment&lt;/strong&gt;</td></tr><tr><td>flexible plastic support &amp;#8212; GelBond PAG Film for Polyacrylamide Gels</td><td>GE Healthcare Bio-Sciences</td><td>309819</td><td></td></tr><tr><td>Glass Plates</td><td>Slumpys</td><td>GBS4100SFSL</td><td></td></tr><tr><td>50 ml conical tubes</td><td>Fisher Scientific</td><td>3181345107</td><td></td></tr><tr><td>15 ml conicals tubes</td><td>FALCON</td><td>352097</td><td></td></tr><tr><td>Micro centrifuge tubes</td><td>Fisher Scientific</td><td>2 ml: 02681258</td><td></td></tr><tr><td>96-well plate (flat bottom)</td><td>Fisher Scientific</td><td>12565501</td><td></td></tr><tr><td>Disposable Pipettes (1&amp;nbsp;ml, 2&amp;nbsp;ml, 5&amp;nbsp;ml, 10&amp;nbsp;ml, 25&amp;nbsp;ml, 50&amp;nbsp;ml)</td><td>Fisher Scientific</td><td>1 ml: 13-678-11B, 2&amp;nbsp;ml: 05214038, 5&amp;nbsp;ml (FALCON): 357529, 10&amp;nbsp;ml: 13-678-11E, 25&amp;nbsp;ml: 13-678-11, 50&amp;nbsp;ml: 13-678-11F</td><td></td></tr><tr><td>Glass Transfer Pipettes</td><td>Fisher Scientific</td><td>5 3/4&quot;: 1367820A, 9&quot;:136786B</td><td></td></tr><tr><td>Pipette Tips (1-200&amp;nbsp;&amp;#956;l, 101-1000&amp;nbsp;&amp;#956;l)</td><td>Fisher Scientific</td><td>2707509</td><td></td></tr><tr><td>Plastic Standard Disposable Transfer Pipettes</td><td>Fisher Scientific</td><td>13-711-9D</td><td></td></tr><tr><td>Parafilm</td><td>PARAFILM&amp;nbsp;</td><td>PM992</td><td></td></tr><tr><td>Powder Free Examination Gloves</td><td>Quest</td><td>92897</td><td></td></tr><tr><td>Silicone spacers &amp;#8212; Silicone sheet, 0.5 mm thick/13 cm x 18 cm</td><td>Grace Bio-Labs</td><td>JTR-S-0.5</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Large/non-disposable lab equipment&lt;/strong&gt;</td></tr><tr><td>Light and Flourescent Microscope (Axiovert 200M)</td><td>Zeiss</td><td>3820005619</td><td></td></tr><tr><td>Microscope Software</td><td>Zeiss</td><td>AxioVision Rel. 4.8.2</td><td></td></tr><tr><td>UV oven</td><td>UVITRON</td><td>UV1080</td><td></td></tr><tr><td>Vacuum chamber/degasser</td><td>BelArt</td><td>999320237</td><td></td></tr><tr><td>Vacuum pump for degasser</td><td>KNF Lab</td><td>5097482</td><td></td></tr><tr><td>Tissue Culture Hood</td><td>NUAIRE</td><td>NU-425-600</td><td></td></tr><tr><td>Chemical Fume Hood</td><td>KEWAUNEE</td><td>99151</td><td></td></tr><tr><td>Inverted Microscope (Axiovert 25)</td><td>Zeiss</td><td>663526</td><td></td></tr><tr><td>Incubator</td><td>NUAIRE</td><td>NU-8500</td><td></td></tr><tr><td>Pipette Aid</td><td>Drummond Scientific Co.</td><td>P-76864</td><td></td></tr><tr><td>Hemacytometer</td><td>Bright-Line</td><td>383684</td><td></td></tr></tbody></table>

simple polyacrylamide-based multiwell stiffness assay for the study of stiffness-dependent cell responses

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a matrix that is elastic and much softer (0.1 &#8211; 100 kPa). Since such stiffness mismatch greatly affects cell responses, there is a strong interest in developing hydrogel materials that span a wide range of stiffness to serve as cell substrates. Polyacrylamide gels, which are inexpensive and cover the stiffness range of all soft tissues in the body, are the hydrogel of choice for many research groups. However, polyacrylamide gel preparation is lengthy, tedious, and only suitable for small batches. Here, we describe an assay which by utilizing a permanent flexible plastic film as a structural support for the gels, enables the preparation of polyacrylamide gels in a multiwell plate format. The technique is faster, more efficient, and less costly than current methods and permits the preparation of gels of custom sizes not otherwise available. As it doesn&#8217;t require any specialized equipment, the method could be easily adopted by any research laboratory and would be particularly useful in research focused on understanding stiffness-dependent cell responses.

聚丙烯酰胺（PA）的水凝胶被广泛用来测试刚性依赖性细胞反应。17,24通过混合不同浓度的丙烯酰胺（A）和双-丙烯酰胺（B），可以使跨越大部分软组织中的刚度范围PA的凝胶主体- 0.3 - 300千帕的杨氏模量1但是，制备的聚丙烯酰胺凝胶的是乏味和耗时的，常常限制了其在"高通量"的应用，例如药物筛选效用12在这里，为简单而快速的方法组装的PA凝胶在多井板或任何其它期望?...

Watch this Scientific Journal Video about Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses at JoVE.com

Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses

Here, a method that enables quick, efficient, and inexpensive preparation of polyacrylamide gels in a multiwell plate format is described. The method does not require any specialized equipment and could be easily adopted by any research laboratory. It would be particularly useful in research focused on understanding stiffness-dependent cell responses.

简单的聚丙烯酰胺为基础的多井刚度检测方法的刚度依赖的细胞反应的研究

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a ...

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JoVE Journal

Bioengineering

19.6K 次观看.  Saint Louis University. Here, a method that enables quick, efficient, and inexpensive preparation of polyacrylamide gels in a multiwell plate format is described. The method does not require any specialized equipment and could be easily adopted by any research laboratory. It would be particularly useful in research focused on understanding stiffness-dependent cell responses.

视频: Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses

Preparation of Complaint Matrices for Quantifying Cellular Contraction

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling. Focal adhesions are macromolecular assemblies that couple the contractile F-actin cytoskeleton to the ECM. This connection allows for the transmission of intracellular mechanical forces across the cell membrane to the underlying substrate. Recent work has shown the mechanical properties of the ECM regulate focal adhesion and F-actin morphology as well as numerous physiological processes, including cell differentiation, division, proliferation and migration. Thus, the use of cell culture substrates has become an increasingly prevalent method to precisely control and modulate ECM mechanical properties.
To quantify traction forces at focal adhesions in an adherent cell, compliant substrates are used in conjunction with high-resolution imaging and computational techniques in a method termed traction force microscopy (TFM). This technique relies on measurements of the local magnitude and direction of substrate deformations induced by cellular contraction. In combination with high-resolution fluorescence microscopy of fluorescently tagged proteins, it is possible to correlate cytoskeletal organization and remodeling with traction forces.
Here we present a detailed experimental protocol for the preparation of two-dimensional, compliant matrices for the purpose of creating a cell culture substrate with a well-characterized, tunable mechanical stiffness, which is suitable for measuring cellular contraction. These protocols include the fabrication of polyacrylamide hydrogels, coating of ECM proteins on such gels, plating cells on gels, and high-resolution confocal microscopy using a perfusion chamber. Additionally, we provide a representative sample of data demonstrating location and magnitude of cellular forces using cited TFM protocols.

In this video, we demonstrate the experimental techniques used to fabricate compliant, extracellular matrix (ECM) coated substrates suitable for cell culture, and which are amenable to traction force microscopy and observing effects of ECM stiffness on cell behavior.

投诉矩阵量化细胞收缩的准备工作

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling.  Focal adhesions are ...

科研

生物工程

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell proliferation, differentiation, migration and cell-cell interactions. The two key parameters of cellular biomechanics are cellular deformability or stiffness and the ability of the cells to contract and generate force. Here we describe a quick and simple method to estimate cell stiffness by measuring the degree of membrane deformation in response to negative pressure applied by a glass micropipette to the cell surface, a technique that is called Micropipette Aspiration or Microaspiration.
Microaspiration is performed by pulling a glass capillary to create a micropipette with a very small tip (2-50 &mu;m diameter depending on the size of a cell or a tissue sample), which is then connected to a pneumatic pressure transducer and brought to a close vicinity of a cell under a microscope. When the tip of the pipette touches a cell, a step of negative pressure is applied to the pipette by the pneumatic pressure transducer generating well-defined pressure on the cell membrane. In response to pressure, the membrane is aspirated into the pipette and progressive membrane deformation or "membrane projection" into the pipette is measured as a function of time. The basic principle of this experimental approach is that the degree of membrane deformation in response to a defined mechanical force is a function of membrane stiffness. The stiffer the membrane is, the slower the rate of membrane deformation and the shorter the steady-state aspiration length.The technique can be performed on isolated cells, both in suspension and substrate-attached, large organelles, and liposomes.
Analysis is performed by comparing maximal membrane deformations achieved under a given pressure for different cell populations or experimental conditions. A "stiffness coefficient" is estimated by plotting the aspirated length of membrane deformation as a function of the applied pressure. Furthermore, the data can be further analyzed to estimate the Young's modulus of the cells (E), the most common parameter to characterize stiffness of materials. It is important to note that plasma membranes of eukaryotic cells can be viewed as a bi-component system where membrane lipid bilayer is underlied by the sub-membrane cytoskeleton and that it is the cytoskeleton that constitutes the mechanical scaffold of the membrane and dominates the deformability of the cellular envelope. This approach, therefore, allows probing the biomechanical properties of the sub-membrane cytoskeleton.

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

基质贴壁细胞的微管吸吮的估计单元刚度

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

使用原子力显微镜测量活细胞的力学性能

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells

Rigid tumor tissues have been strongly implicated in regulating cancer cell migration and invasion. Invasive migration through cross-linked tissues is facilitated by actin-rich protrusions called invadopodia that proteolytically degrade the extracellular matrix (ECM). Invadopodia activity has been shown to be dependent on ECM rigidity and cancer cell contractile forces suggesting that rigidity signals can regulate these subcellular structures through actomyosin contractility. Invasive and contractile properties of cancer cells can be correlated in vitro using invadopodia and traction force assays based on polyacrylamide gels (PAAs) of different rigidities. Invasive and contractile properties of cancer cells can be correlated in vitro using invadopodia and traction force assays based on polyacrylamide gels (PAAs) of different rigidities. While some variations between the two assays exist, the protocol presented here provides a method for creating PAAs that can be used in both assays and are easily adaptable to the user&#8217;s specific biological and technical needs.

Mechanical rigidity in the tumor microenvironment plays a crucial role in driving malignant behavior by increasing invadopodia activity and actomyosin contractility. Using polyacrylamide gels (PAAs), invadopodia and traction force assays can be utilized to study the invasive and contractile properties of cancer cells in response to matrix rigidity.

聚丙烯酰胺凝胶侵袭伪足和牵引力化验的肿瘤细胞

Rigid tumor tissues have been strongly implicated in regulating cancer cell migration and invasion. Invasive migration through cross-linked tissues is ...

医学

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically12. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.
The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution12. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) &lt;3000 Pascal and stiff substrata/tissues as those with E &gt;20,000 Pascal.

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

使用丙烯酰胺水凝胶的细胞功能的研究矩阵刚度的影响

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

生物学

An Optimized O9-1/Hydrogel System for Studying Mechanical Signals in Neural Crest Cells

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to various organs and tissues. Tissue stiffness produces mechanical force, a physical cue that plays a critical role in NCC differentiation; however, the mechanism remains unclear. The method described here provides detailed information for the optimized generation of polyacrylamide hydrogels of varying stiffness, the accurate measurement of such stiffness, and the evaluation of the impact of mechanical signals in O9-1 cells, a NCC line that mimics in vivo NCCs.
Hydrogel stiffness was measured using atomic force microscopy (AFM) and indicated different stiffness levels accordingly. O9-1 NCCs cultured on hydrogels of varying stiffness showed different cell morphology and gene expression of stress fibers, which indicated varying biological effects caused by mechanical signal changes. Moreover, this established that varying the hydrogel stiffness resulted in an efficient in vitro system to manipulate mechanical signaling by altering gel stiffness and analyzing the molecular and genetic regulation in NCCs. O9-1 NCCs can differentiate into a wide range of cell types under the influence of the corresponding differentiation media, and it is convenient to manipulate chemical signals in vitro. Therefore, this in vitro system is a powerful tool to study the role of mechanical signaling in NCCs and its interaction with chemical signals, which will help researchers better understand the molecular and genetic mechanisms of neural crest development and diseases.

Detailed step-by-step protocols are described here for studying mechanical signals in vitro using multipotent O9-1 neural crest cells and polyacrylamide hydrogels of varying stiffness.

一种优化的O9-1/水凝胶系统，用于研究神经嵴细胞中的机械信号

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to ...

发育生物学

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been proven to be useful substrates for culturing cells in a physical microenvironment that partially mimics in vivo conditions. Here, we present a simple protocol for characterizing the Young's moduli of isotropic linear elastic substrates typically used for mechanobiology studies. The protocol consists of preparing a soft silicone substrate on a Petri dish or stiff silicone, coating the top surface of the silicone substrate with fluorescent beads, using a millimeter-scale sphere to indent the top surface (by gravity), imaging the fluorescent beads on the indented silicone surface using a fluorescence microscope, and analyzing the resultant images to calculate the Young's modulus of the silicone substrate. Coupling the substrate's top surface with a moduli extracellular matrix protein (in addition to the fluorescent beads) allows the silicone substrate to be readily used for cell plating and subsequent studies using traction force microscopy experiments. The use of stiff silicone, instead of a Petri dish, as the base of the soft silicone, enables the use of mechanobiology studies involving external stretch. A specific advantage of this protocol is that a widefield fluorescence microscope, which is commonly available in many labs, is the major equipment necessary for this procedure. We demonstrate this protocol by measuring the Young's modulus of soft silicone substrates of different elastic moduli.

Substrates with stiffness in the kilopascal-range are useful to study the response of cells to physiologically relevant micro-environment stiffness. Using just a widefield fluorescence microscope, the Young's modulus of soft silicone gels can be determined using an indentation with a suitable sphere.

用 Widefield 荧光显微镜研究软硅基美国匹茨堡的刚度测量

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been ...

A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, function, and fate in general. Although increasingly more adherent cell types&#39; responses to matrix stiffness have been characterized, how adherent cells&#39; susceptibility to bacterial infection depends on matrix stiffness is largely unknown, as is the effect of bacterial infection on the biomechanics of host cells. We hypothesize that the susceptibility of host endothelial cells to a bacterial infection depends on the stiffness of the matrix on which these cells reside, and that the infection of the host cells with bacteria will change their biomechanics. To test these two hypotheses, endothelial cells were used as model hosts and Listeria monocytogenes as a model pathogen. By developing a novel multi-well format assay, we show that the effect of matrix stiffness on infection of endothelial cells by L. monocytogenes can be quantitatively assessed through flow cytometry and immunostaining followed by microscopy. In addition, using traction force microscopy, the effect of L. monocytogenes infection on host endothelial cell biomechanics can be studied. The proposed method allows for the analysis of the effect of tissue-relevant mechanics on bacterial infection of adherent cells, which is a critical step towards understanding the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria. This method is also applicable to a wide variety of other types of studies on cell biomechanics and response to substrate stiffness where it is important to be able to perform many replicates in parallel in each experiment.

We have developed a multi-well format polyacrylamide-based assay for probing the effect of extracellular matrix stiffness on bacterial infection of adherent cells. This assay is compatible with flow cytometry, immunostaining, and traction force microscopy, allowing for quantitative measurements of the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria.

基于聚丙烯酰胺的多井格式实验研究细胞外基质硬度对黏附细胞细菌感染的影响

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, ...

免疫与感染

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 ...

Quantitative Time-Lapse Microscopy for Assessing Extracellular Matrix Stiffness-Dependent Bacterial Dissemination

Source: Bastounis, E. E. et al., <a href="https://app.jove.com/v/57361">A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells.</a> J. Vis. Exp.&#160;(2018)
This video showcases quantitative time-lapse microscopy for evaluating extracellular matrix stiffness-dependent Listeria monocytogenes dissemination through endothelial cells. A softer hydrogel facilitates faster bacterial dissemination through endothelial cells compared to stiffer counterparts, highlighting the influence of matrix stiffness on bacterial dissemination.

用于评估细胞外基质刚度依赖性细菌传播的定量延时显微镜

1. Manufacturing Thin Two-layered Polyacrylamide (PA) Hydrogels on Multi-Well Plates

	Dissolve ammonium persulfate (APS) in distilled ultrapure water to ...