我们感谢乌尔里希施瓦茨实验室用于蜂窝牵引力（Sabass 等 ，2008）的量化的计算跟踪软件。这项工作是由宝来惠康事业奖和美国国立卫生研究院主任的先锋奖（DP10D00354）ML Gardel和医学科学家的国家研究服务奖（5 T32 GM07281）到SP冬季的支持。

1。激活盖玻片表面<ol><li>盖玻片（＃1.5，22x40毫米）清洁使用在前面描述的协议（沃特曼斯托勒，1998年）的一系列肥皂和乙醇洗涤清洁和去除灰尘。 </li><li>放置在一个不锈钢架机架盖玻片，这样盖玻片间距不接触。 </li><li>在化学通风柜（丁腈手套和护目镜建议），终浓度为2％，在异丙醇稀释充满力量3 aminopropyltrimethoxysilane（2毫升硅烷/ 100毫升异丙醇），以填补一个正方形的玻璃盘（〜350毫升量）。由于塑料的反应，使用玻璃巴斯德吸管申请3 aminopropyltrimethoxysilane异丙醇。 </li><li>完全沉浸盖玻片从1.2到这10分钟的解决方案，同时轻轻搅拌在搅拌台通风柜。 </li><li>沉浸在DDH 2 O（4水交换）清洗盖玻片。允许浸泡10分钟，最终交换的时间，搅拌，。含氨基硅烷解决方案应作为危险废物处置。 </li><li>干盖玻片，在温暖的温度在孵化器（〜37 ° C）在无尘的环境中10分钟。 </li><li>冷却至室温。 </li><li>在通风柜中，浸泡在1％的戊二醛溶液在玻璃方盘 2 DDH的盖玻片上搅拌30分钟盘。 </li><li>洗为10分钟，每交换3 DDH 2 O的交流，并搅拌。戊二醛作为危险废物处置。 </li><li>在室温下干燥，用铝箔覆盖，以避免灰尘粘的盖玻片。 </li><li>储存在干燥的地方，远离灰尘，长达2个月。 </li></ol> 2。制备聚丙烯酰胺（PAA）凝胶<ol><li>准备从40％的丙烯酰胺和双丙烯酰胺，2％以下表1丙烯酰胺/双丙烯酰胺组合的股票解决方案。我们坚持几个股票，不同刚度PAA凝胶优化的解决方案;在表1和表2中列出的例子。联合解决方案可以保持好几年了，只要他​​们在黑暗的瓶保持在4℃ </li><li>从库存解决方案，获得工作的解决方案，包含最终所需浓度的丙烯酰胺/双丙烯酰胺。例如，我们准备了7.5％acrylamide/0.10％双丙烯酰胺在DDH 2 O的工作解决方案，为2.8kPa PAA凝胶。 </li><li>德加在20分钟的真空室中的丙烯酰胺溶液，以减少氧气的解决方案，它可以防止临机聚合内。 </li><li>准备10％过硫酸铵（APS）解决方案（0.5g/5mL）。使用新的工作在3天之内股票。另外，股票被冻结要在以后的日期使用。 </li><li>尽管丙烯酰胺是脱气，雨- X的抹布擦拭一个1X3的“显微镜载玻片大力载玻片表面的疏水要除去多余的雨 - X，​​设置玻璃擦拭玻璃用湿布Kimwipe幻灯片。幻灯片一边，有盖。 </li><li>从真空室中取出丙烯酰胺溶液，添加荧光珠（量的1％，表1中列出的工作液5μL）。加入0.75μlTEMED和2.5μLAPS的10％，这将启动凝胶聚合。 〜5秒移液拌匀，以尽量减少引入的气泡， </li><li>应用10-12μl丙烯酰胺溶液的疏水性显微镜幻灯片（在步骤2.4准备）和就地激活液滴顶部22x40mm盖玻片的。凝胶溶液应大衣整个盖玻片。平滑内解决可能出现的任何气泡。允许的凝胶溶液聚合在室温〜10分钟。 </li><li>聚合完成后，可以评估反相剩余工作的离心管中的解决方案。此外，聚合凝胶可拉离盖玻片边缘。宏观聚合后立即观察，独立的盖玻片从载玻片上。使用精尖的镊子或刀片边缘，小心地取出盖玻片，用凝胶附着，从显微镜玻片表面浸入凝胶和DDH 2，保持水化。 </li></ol> 3。 PAA凝胶耦合细胞外基质（ECM）的蛋白质三种不同的方法可用于附加的ECM蛋白的PAA凝胶的顶部表面（3.1和3.2），或含有凝胶体积（3.3）内的ECM蛋白。在这里，我们讨论PAA凝胶纤维连接蛋白的耦合，导致孵化后玻璃上的1小时10微克/毫升纤维连接蛋白溶液吸附量相当于一个表面配基密度。选择方法的注意事项有详细的讨论。 <ol><li>交联对蛋白质的活性胺磺酸基SANPAH ECM蛋白PAA凝胶表面共价连接到PAA凝胶表面的异型交联剂氨基SANPAH <ol><li>准备解散小号40μL氨基SANPAH工作等分<html> ulfo SANPAH粉在无水二甲基亚砜（DMSO）（20μL每毫克氨基SANPAH）。闪存冻结的股票在液氮储存于-80 ° C供以后使用。 </li><li>取出凝胶表面使用盖玻片微调（&lt;2秒）DDH 2 O。避免干燥的凝胶。 </li><li> DDH 2 O（2mg/ml，pH值7）使用前和大衣凝胶表面（约200μL）稀释磺酸基SANPAH二甲基亚砜等分。请注意，磺酸基SANPAH反应半衰期短（约5分钟）在室温下在水中，因此，这些措施应以迅猛的速度进行。 </li><li>凝胶表面暴露在紫外线照射下，在紫外线的交联剂烤箱（8W，254 nm波长在2-3英寸1.5分钟的距离）。磺酸基SANPAH会改变颜色从橙色至褐色。 </li><li>在一个烧杯用新鲜的DDH 2 O的DIP紫外线处理过的盖玻片和删除多余的水从凝胶表面使用盖玻片微调（&lt;2秒） 。 </li><li>移液器〜50μL上的封口膜冷1mg/ml的纤维连接蛋白（FN）（PBS，pH值7.4），在培养皿容器。反转盖玻片上的FN下降之上，凝胶方面暴露新生力量。 </li><li>在室温下反应1-2小时或4℃过夜。 </li><li>在6厘米的组织文化，包含PBS（pH 7.4）中菜放置盖玻片，足以覆盖盖玻片，在组织培养罩中的无菌条件下。 </li><li>广泛与几个洗（3-5）的PBS液（pH 7.4），在无菌条件下清洗。 </li><li>在组织培养罩中使用30分钟杀菌灯消毒盖玻片。 </li><li>细胞介质中孵育30-45分钟前电镀细胞的盖玻片。 </li></ol></li><li>交叉连接流脑水合肼碳水化合物对蛋白质组的临时机场管理局凝胶表面被氧化和耦合凝胶使用水合肼。 <ol><li>准备第2条所述的聚丙烯酰胺凝胶。 </li><li> PAA凝胶在塑料培养皿中，在通风柜和使用手套的盖玻片放置吸管上至少两个小时，每个PAA凝胶和孵化表面约1毫升未经稀释的水合肼，但最长不超过24小时</li><li>添加DDH 2 O的培养皿中;删除水合肼解决方案和处置危险废物。 </li><li>培养皿中添加5％的醋酸浸泡盖玻片。封面和孵育1小时。 </li><li>删除乙酸和洗DDH 2 O在DDH 2 O的孵育一小时。现在激活的盖玻片和交叉链接氧化纤维连接蛋白（FN）。 </li><li>稀释10μL为1 mg / ml的FN溶液940μL的50 mM的醋酸钠缓冲液（pH值4.5）在一个黑暗的微离心管中，使终浓度为10微克/毫升。 </li><li> 50毫米醋酸钠缓冲液（pH值4.5），以1毫升加入80毫克的钠元碘酸20X元，高碘酸钠的股票。 </li><li> 3.2.6准备加入50μL20X钠元碘酸股票的FN解决方案，这样，最终工作浓度是10微克/毫升FN和4微克/毫升钠元碘酸。在室温下孵育30分钟，在黑暗中管。 </li><li> 3.2.5准备使用盖玻片微调（&lt;2秒）的表面活化凝胶，删除多余的DDH 2 O。避免干燥的凝胶。 </li><li>吸取〜500μLFN到激活凝胶表面，在室温下1小时的孵化解决方案。 </li><li>放置盖玻片的菜肴，含PBS液（pH 7.4），足以覆盖盖玻片。 </li><li> （3-5）的PBS（pH 7.4）中的几个洗洗净广泛。 </li><li>在组织培养罩中使用30分钟杀菌灯消毒盖玻片。 </li><li>细胞介质中孵育30-45分钟前电镀细胞的盖玻片。 </li></ol></li><li> ECM PAA凝胶蛋白丙烯- X的散装琥珀酰亚胺酯共轭，该协议是前2.5。对蛋白质的活性胺耦合与NHS酯化学丙烯酰胺单体，然后聚合成批量的PAA凝胶。 <ol><li>共轭ECM蛋白的丙烯- X的每制造商的说明选择。共轭蛋白联合解决方案应保存在4 ° C。 </li><li>凝胶制造（如10％盖玻片UL）所需的临时机场管理局的工作解决方案的计算量。 </li><li>减水从表1中的工作液配方上市量50μL（例如10％的体积），并引发聚合。 </li><li>删除3.3.2计算的体积和10％的体积增加ECM​​ /丙烯- X的解决方案。 （如1μLECM /丙烯 - X 9μL的PAA溶液）。这一步应该是执行迅速，凝胶聚合。 </li><li>如前所述，2.6和2.7的完整步骤。 </li><li>广泛与几个洗（3-5）的PBS液（pH 7.4），在无菌条件下清洗。 </li><li>在组织培养罩中使用30分钟杀菌灯消毒盖玻片。 </li><li>细胞介质中孵育30-45分钟前电镀细胞的盖玻片。 </li></ol></li></ol>乐“&gt; 4。载入盖玻片到共焦成像室这些步骤进行后，细胞已被允许传播ECM涂层凝胶基片（约6-12小时）。要组装的共焦成像室（RC - 30WA），它是有用的咨询指导华纳仪器网站。 <ol><li>温暖的细胞介质和0.5％或0.25％胰蛋白酶和成5毫升或10ml注射器的负载。 </li><li>加载到华纳仪器共​​焦成像室（RC - 30WA）持有热门盖玻片22x30mm盖玻片，用真空油脂，以保持在盖玻片。 </li><li>将一腔形成盖玻片上的橡胶垫片，允许访问入口和出口聚乙烯管材。这将使顶部盖玻片和凝胶涂层22x40mm盖玻片，垫片的大小而定150-1000微米之间的间距。 </li><li>负载入口管注射器上，通过连接器套件，并检查，媒体流通过油管和到顶部盖玻片，无气泡，流线明显。 </li><li>应用到基地的商会和负载凝胶涂22x40mm盖玻片，细胞面朝上的真空油脂。温暖媒体应用细胞。 </li><li>放置到腔基地顶部盖玻片持有人，商会垫片分离的凝胶涂层的盖玻片。确保坐在内室基地的定位销定位孔在顶端盖玻片。 </li><li>应用压型钢板室基地，并使用螺丝和安全的压型钢板压型钢板扳手。 </li><li>检查通过油管和商会的流媒体监控室中的任何潜在的泄漏和消除细胞表面上的的任何媒体自由贸易区。注：用一个小针头注入画一个轻微的真空，同时与媒体，可以帮助消除媒体自由区。 </li><li>应用共焦成像室内成像显微镜持有人的舞台适配器。 </li><li>图片荧光标记蛋白和共聚焦荧光显微镜凝胶基材内嵌入的荧光珠。 </li><li>要获得的凝胶内无应变珠职位的形象，灌注胰蛋白酶从凝胶中分离的细胞粘连，和采取的，在相同的成像领域，坚持细胞的荧光珠的形象。比较紧张，无应变珠立场允许下收缩凝胶基材位移量化。 </li></ol>代表性的成果： 上述协议描述为准备符合PAA凝胶研究细胞收缩的实验过程，如图1所示。与本协议获得的凝胶表面相对平整，光滑，嵌入式均匀的荧光珠（图2A）。 如果在粘着斑的位置，细胞成像（图3A）和凝胶表面（图3B）测量凝胶收缩应该做的共焦光学平面粘着斑。凝胶的收缩，可在凝胶表面的可视化，当细胞贴壁（紧张）与分离（非应变）嵌入式荧光灯珠的位移（图3B）。计算算法的使用，可以产生与凝胶珠的位移和相应的弹性模量（图3C和3D）（Sabass等，2008 ）的牵引强调。如果成像发生在凝胶深，然后将珠的位移较小，不粘着斑产生的牵引力量的代表。 <img src="/files/ftp_upload/2173/2173fig1.jpg" alt="图1" /> 图1实验装置示意图。此过程的总体目标是创建兼容的矩阵为研究细胞收缩的目的。实验过程的第一步是激活amino-silane/glutaraldehyde治疗为目的的锚固聚合凝胶的盖玻片。第二步是聚合聚丙烯酰胺凝胶，含有荧光珠，到激活盖玻片。第三步涉及的化学交叉连接胞外配体的聚丙烯酰胺凝胶表面，使用第3步中列出的三个耦合技术之一。细胞，然后镀上凝胶，坚持和传播。根据活跃的细胞收缩，嵌入在凝胶珠取代。 <img src="/files/ftp_upload/2173/2173fig2.jpg" alt="图2" /> 图2。PAA凝胶的顶部表面的光学共焦片，可视化（A）内凝胶荧光的40nm嵌入式珠和纤维连接蛋白（B）免疫。 <img src="/files/ftp_upload/2173/2173fig3.jpg" alt="图3" /> 图3。牵引力实验代表性的结果。 （A）在人成骨肉瘤U2OS细胞的粘着斑是Marked GFP - Paxillin的嵌入在PAA底层紧张（绿色）和非应变（红色）的重点粘连凝胶的荧光珠（二）岗位。箭头指示珠位移的例子。 （三）牵引应力向量和（四）相应的热规模牵引地图强调来自凝胶收缩，采用计算算法（Sabass 等 ，2008）。比例尺= 5微米。 表1： 范例股票和工作的临机解决方案（表1中的数据是首次获得杨等人。，并独立在我们的实验室证实。） <table border="1"><tbody><tr><td colspan="5"> 股票临时机场管理局的解决方案 </td></tr><tr><td> 剪切模量（PA）的PAA凝胶 </td><td> 230 </td><td> 2833 </td><td> 8640 </td><td> 16344 </td></tr><tr><td> 40％丙烯酰胺（毫升） </td><td> 1.25 </td><td> 3.12 </td><td> 2.34 </td><td> 2.50 </td></tr><tr><td> 2％双丙烯酰胺（毫升） </td><td> 0.50 </td><td> 0.83 </td><td> 1.88 </td><td> 0.60 </td></tr><tr><td> 水（毫升） </td><td> 3.25 </td><td> 1.04 </td><td> 0.78 </td><td> 1。 90 </td></tr><tr><td> 总体积（ml）： </td><td> 5 </td><td> 5 </td><td> 5 </td><td> 5 </td></tr></tbody></table> <table border="1"><tbody><tr><td colspan="5"> 工作临机解决方案</td></tr><tr><td> 联合解决方案中使用（PA） </td><td> 230 </td><td> 2833 </td><td> 8640 </td><td> 16344 </td></tr><tr><td> 股票溶液的体积（微升） </td><td> 150 </td><td> 150 </td><td> 200 </td><td> 300 </td></tr><tr><td> 水（微升） </td><td> 341.75 </td><td> 341.75 </td><td> 291.75 </td><td> 191.75 </td></tr><tr><td> 珠（微升） </td><td> 5 </td><td> 5 </td><td> 5 </td><td> 5 </td></tr><tr><td> TEMED（微升） </td><td> 0.75 </td><td> 0.75 </td><td> 0.75 </td><td> 0.75 </td></tr><tr><td> 10％的APS（微升） </td><td> 2.5 </td><td> 2.5 </td><td> 2.5 </td><td> 2.5 </td></tr><tr><td> 总体积（μL）： </td><td> 500 </td><td> 500 </td><td> 500 </td><td> 500 </td></tr><tr><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 最后丙烯酰胺％ </td><td> 3 </td><td> 7.5 </td><td> 7.5 </td><td> 12 </td></tr><tr><td> 最终双丙烯酰胺％ </td><td> 0.06 </td><td> 0.1 </td><td> 0.3 </td><td> 0.15 </td></tr></tbody></table> 表2： 剪切模量的PAA基板的各种最终丙烯酰胺和双丙烯酰胺的百分比<table border="1"><tbody><tr><td colspan="2"> 12％丙烯酰胺 </td><td></td><td colspan="2"> 7.5％丙烯酰胺 </td></tr><tr><td> ％双丙烯酰胺</td><td>剪切模量（PA） </td><td></td><td> ％双丙烯酰胺</td><td>剪切模量（PA） </td></tr><tr><td> 0.145 </td><td> 16344 </td><td></td><td> 0.01 </td><td> 689 </td></tr><tr><td> 0.28 </td><td> 30067 </td><td></td><td> 0.03 </td><td> 1535 </td></tr><tr><td> 0.45 </td><td> 34263 </td><td></td><td> 0.05 </td><td> 2286 </td></tr><tr><td> 0.55 </td><td> 42375 </td><td></td><td> 0.075 </td><td> 2833 </td></tr><tr><td> 0.575 </td><td> 50873 </td><td></td><td> 0.1 </td><td> 4069 </td></tr><tr><td> 0.6 </td><td> 55293 </td><td></td><td> 0.2 </td><td> 5356 </td></tr><tr><td></td><td></td><td></td><td> 0.3 </td><td> 8640 </td></tr><tr><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="2"> 5％丙烯酰胺 </td><td></td><td colspan="2"> 3％丙烯酰胺 </td></tr><tr><td> ％双丙烯酰胺</td><td>剪切模量（PA） </td><td></td><td> ％双丙烯酰胺</td><td>剪切模量（PA） </td></tr><tr><td> 0.05 </td><td> 430 </td><td></td><td>0.02 </td><td> 1.3 </td></tr><tr><td> 0.075 </td><td> 600 </td><td></td><td> 0.04 </td><td> 54 </td></tr><tr><td> 0.1 </td><td> 1431 </td><td></td><td></td><td></td></tr></tbody></table>

<ol>
	<li>Damljanovic, V., Lajerholm, B. C., Jacobson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bulk+and+micropatterned+conjugation+of+extracellular+matrix+proteins+to+characterized+polyacrylamid+substrates+for+cell+mechanotransduction+assays.">Bulk and micropatterned conjugation of extracellular matrix proteins to characterized polyacrylamid substrates for cell mechanotransduction assays.</a> Biotechniques. 39 (6), 847-851 (2005).</li><li>Engler, A., Bacakova, L. N. e. w. m. a. n., Hategan, C., Griffin, A., M, D. D. i. s. c. h. e. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate+compliance+versus+ligand+in+cell+on+gel+responses.">Substrate compliance versus ligand in cell on gel responses.</a> Biophys J. 86 ((1 Pt 1)), 617-628 (2004).</li><li>Gardel, M. L., Sabass, B., Ji, L., Danuser, G., Schwarz, U. S., Waterman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traction+stress+in+focal+adhesions+correlates+biphasically+with+actin+retrograde+flow+speed.">Traction stress in focal adhesions correlates biphasically with actin retrograde flow speed.</a> J Cell Biol. 183, 999-1005 (2008).</li><li>Rajagopalan, P., Marganski, W. A., Brown, X. Q., Wong, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+comparison+of+the+spread+area,+contractility,+and+migration+of+balb/c+3T3+fibroblasts+adhered+to+fibronectin-+and+RGD-modified+substrata.">Direct comparison of the spread area, contractility, and migration of balb/c 3T3 fibroblasts adhered to fibronectin- and RGD-modified substrata.</a> Biophys J. 87 (4), 2818-2827 (2004).</li><li>Reinhart-King, C. A., Dembo, M., Hammer, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamics+and+mechanics+of+endothelial+cell+spreading.">The dynamics and mechanics of endothelial cell spreading.</a> Biophys J. 89, 676-689 (2005).</li><li>Stricker, J., Sabass, B., Schwarz, U. S., Gardel, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+traction+force+microscopy+for+micron-sized+focal+adhesions.">Optimization of traction force microscopy for micron-sized focal adhesions.</a> J. Phys: Condensed Matter. 22, 194104-194114 (2010).</li><li>Sabass, B., Gardel, M. L., Waterman, C. M., Schwarz, U. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+traction+force+microscopy+based+on+experimental+and+computational+advances.">High resolution traction force microscopy based on experimental and computational advances.</a> Biophys J. 94, 207-220 (2008).</li><li>Yeung, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+substrate+stiffness+on+cell+morphology,+cytoskeletal+structure,+and+adhesion.">Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion.</a> Cell Motil Cytoskeleton. 60 (1), 24-34 (2005).</li><li>Waterman-Storer, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule/organelle+motility+assays.">Microtubule/organelle motility assays.</a> Curr Protoc Cell Biol. , 13.1.1-13.1.21 (1998).</li></ol>

这里描述的步骤设置牵引力显微镜（TFM）实验，计算跟踪程序的实施等，2008）（Sabass，可量化与微米级的空间分辨率的细胞力量。为了优化实验方案，关键是形成一个纯粹的和统一的凝胶基板与ECM的配体的镀层均匀。我们将讨论下面潜在的隐患： 非均匀的凝胶表面或眼泪： 在我们的经验，有几个步骤，似乎形成了一个漂亮的制服凝胶的关键。如果?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<tr>
<td>3-aminopropyltrimethyoxysilane</td>
<td>&nbsp;</td>
<td>Aldrich</td>
<td>28, 177-8</td>
<td>&nbsp;</td>
<tr>
<td>40% Acrylamide</td>
<td>&nbsp;</td>
<td>BioRad</td>
<td>161-0140</td>
<td>&nbsp;</td>
<tr>
<td>2% Bis-acrylamide</td>
<td>&nbsp;</td>
<td>Fisher BioReagents</td>
<td>BP1404</td>
<td>&nbsp;</td>
<tr>
<td>TEMED</td>
<td>&nbsp;</td>
<td>Fisher BioReagents</td>
<td>BP 150-20</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium persulfate</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP179</td>
<td>&nbsp;</td>
<tr>
<td>40nm fluorescent micro-spheres</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>F8789</td>
<td>&nbsp;</td>
<tr>
<td>Sulfo-SANPAH</td>
<td>&nbsp;</td>
<td>Pierce</td>
<td>22589</td>
<td>&nbsp;</td>
<tr>
<td>Confocal imaging chamber (RC-30)</td>
<td>&nbsp;</td>
<td>Warner Instruments</td>
<td>64-0320</td>
<td>&nbsp;</td>
<tr>
<td>Coverslip spinner</td>
<td>&nbsp;</td>
<td>Home-built</td>
<td>NA</td>
<td>&nbsp;</td>
<tr>
<td>Ultraviolet lamp CL1000</td>
<td>&nbsp;</td>
<td>UVP</td>
<td>95-0228-01</td>
<td>&nbsp;</td>
<tr>
<td>Stainless steel rack</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>72239-04</td>
<td>&nbsp;</td>
<tr>
<td>acryloyl-X, SE (6-((acryloyl)amino)hexanoic acid)</td>
<td>&nbsp;</td>
<td>Invitrogen
</td>
<td>A-20770</td>
<td>&nbsp;</td>
<tr>
<td>Hydrazine hydrate</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>225819</td>
<td>&nbsp;</td>
<tr>
<td>Sodium meta-periodate</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>20504</td>
<td>&nbsp;</td>
<tr>
<td>Isopropanol</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>A416-4</td>
<td>&nbsp;</td>
<tr>
<td>Fibronectin</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>F2006</td>
<td>&nbsp;</td>
<tr>
<td>Collagen</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>354236</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips (#1.5)</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>2940&#x2010;224</td>
<td>&nbsp;</td>
<tr>
<td>Glutaraldehyde</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>16120</td>
<td>&nbsp;</td>
<tr>
<td>Rain-X</td>
<td>&nbsp;</td>
<td>SOPUS Products</td>
<td>www.rainx.com</td>
<td>&nbsp;</td>
<tr>
<td>Acetic Acid</td>
<td>&nbsp;</td>
<td>Acros Organics</td>
<td>64-19-7</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

preparation of complaint matrices for quantifying cellular contraction

调节细胞粘附到细胞外基质（ECM）是细胞迁移和ECM重构是必要的。粘着斑大分子集会，情侣收缩的F -肌动蛋白细胞骨架的ECM。此连接可以穿过细胞膜，细胞内的机械力的传输底层基板。最近的工作表明ECM的力学性能调节粘着和F -肌动蛋白的形态以及众多的生理过程，包括细胞分化，分裂，增殖和迁移。因此，细胞培养基质的使用，已成为越来越普遍的方法，能够精​​确地控制和调节ECM的力学性能。 为了量化在粘着斑贴壁细胞的牵引力量，兼容基板结合高分辨率成像和计算技术中的一个方法被称为牵引力显微镜（TFM）。这种技术依赖于当地的基板变形诱发细胞收缩幅度和方向的测量。高分辨率荧光显微镜观察荧光标记的蛋白质相结合，这是可能的关联骨架组织和重塑与牵引力。 在这里，我们提出了一个详细的实验方案编制二维的兼容矩阵，为创造一个良好的特点，可调谐的机械刚度，这是适合测量细胞收缩的细胞培养基材的目的。这些协议包括聚丙烯酰胺凝胶的制备，ECM蛋白等凝胶涂料，电镀细胞凝胶，和高分辨率的共聚焦显微镜使用灌注室。此外，我们提供了一个代表性的样本数据表明使用列举的水杉协议的蜂窝力量的位置和震级。

Watch this Scientific Journal Video about Preparation of Complaint Matrices for Quantifying Cellular Contraction at JoVE.com

Preparation of Complaint Matrices for Quantifying Cellular Contraction

在这段视频中，我们展示了用于制造兼容，细胞外基质（ECM）适用于细胞培养镀膜基片的实验技术，适合牵引力显微镜观察细胞的行为流脑刚度的影响。

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 ...

preparation-of-complaint-matrices-for-quantifying-cellular-contraction

Research

JoVE Journal

Bioengineering

17.7K 次观看.  University of Chicago. 在这段视频中，我们展示了用于制造兼容，细胞外基质（ECM）适用于细胞培养镀膜基片的实验技术，适合牵引力显微镜观察细胞的行为流脑刚度的影响。

视频: Preparation of Complaint Matrices for Quantifying Cellular Contraction

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

含有金纳米棒蜂窝车辆的制备及光声分析

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

科研

生物工程

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

The only definitive therapy for end stage heart failure is orthotopic heart transplantation. Each year, it is estimated that more than 100,000 donor hearts are needed for cardiac transplantation procedures in the United States1-2. Due to the limited numbers of donors, only approximately 2,400 transplants are performed each year in the U.S.2. Numerous approaches, from cell therapy studies to implantation of mechanical assist devices, have been undertaken, either alone or in combination, in an attempt to coax the heart to repair itself or to rest the failing heart3. In spite of these efforts, ventricular assist devices are still largely used for the purpose of bridging to transplantation and the utility of cell therapies, while they hold some curative promise, is still limited to clinical trials. Additionally, direct xenotransplantation has been attempted but success has been limited due to immune rejection. Clearly, another strategy is required to produce additional organs for transplantation and, ideally, these organs would be autologous so as to avoid the complications associated with rejection and lifetime immunosuppression. Decellularization is a process of removing resident cells from tissues to expose the native extracellular matrix (ECM) or scaffold. Perfusion decellularization offers complete preservation of the three dimensional structure of the tissue, while leaving the bulk of the mechanical properties of the tissue intact4. These scaffolds can be utilized for repopulation with healthy cells to generate research models and, possibly, much needed organs for transplantation. We have exposed the scaffolds from neonatal mice (P3), known to retain remarkable cardiac regenerative capabilities,5-8 to detergent mediated decellularization and we repopulated these scaffolds with murine cardiac cells. These studies support the feasibility of engineering a neonatal heart construct. They further allow for the investigation as to whether the ECM of early postnatal hearts may harbor cues that will result in improved recellularization strategies.

In these studies, we provide methodology for novel, neonatal, murine cardiac scaffolds for use in regenerative studies.

新生儿心脏支架：再生研究新矩阵

The only definitive therapy for end stage heart failure is orthotopic heart transplantation.  Each year, it is estimated that more than 100,000 donor ...

2D and 3D Matrices to Study Linear Invadosome Formation and Activity

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor cells have to cross anatomical barriers to invade and migrate through the surrounding tissue in order to reach blood or lymphatic vessels. This requires the interaction between cells and the extracellular matrix (ECM). At the cellular level, many cells, including the majority of cancer cells, are able to form invadosomes, which are F-actin-based structures capable of degrading ECM. Invadosomes are protrusive actin structures that recruit and activate matrix metalloproteinases (MMPs). The molecular composition, density, organization, and stiffness of the ECM are crucial in regulating invadosome formation and activation. In vitro, a gelatin assay is the standard assay used to observe and quantify invadosome degradation activity. However, gelatin, which is denatured collagen I, is not a physiological matrix element. A novel assay using type I collagen fibrils was developed and used to demonstrate that this physiological matrix is a potent inducer of invadosomes. Invadosomes that form along the collagen fibrils are known as linear invadosomes due to their linear organization on the fibers. Moreover, molecular analysis of linear invadosomes showed that the discoidin domain receptor 1 (DDR1) is the receptor involved in their formation. These data clearly demonstrate the importance of using a physiologically relevant matrix in order to understand the complex interactions between cells and the ECM.

This protocol describes how to prepare a 2D mixed matrix, consisting of gelatin and collagen I, and a 3D collagen I plug to study linear invadosomes. These protocols allow for the study of linear invadosome formation, matrix degradation activity, and the invasion capabilities of primary cells and cancer cell lines.

2D和3D矩阵研究线性侵袭体形成和活性

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

人工程心脏组织的收缩自动分析心脏药物安全筛选

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.

This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis.

3D 基质中哺乳动物细胞分裂的定量共焦反射显微镜观察

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the standard protocols for generating genomic or transcriptomic libraries are incompatible and researchers must choose whether to sequence DNA or RNA for a particular sample. Gel-seq solves this problem by enabling researchers to simultaneously prepare libraries for both DNA and RNA starting with 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries. We designed Gel-seq so that it could be easily implemented by other researchers; many genetics labs already have the necessary equipment to reproduce the Gel-seq device fabrication. Our protocol employs commonly-used kits for both whole-transcript amplification (WTA) and library preparation, which are also likely to be familiar to researchers already versed in generating genomic and transcriptomic libraries. Our approach allows researchers to bring to bear the power of both DNA and RNA sequencing on a single sample without splitting and with negligible added cost.

Gel-seq enables researchers to simultaneously prepare libraries for both DNA- and RNA-seq at negligible added cost starting from 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries.

凝胶序列: 一种同时测序库的方法用水凝胶基质制备 DNA 和 RNA

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

多路人工细胞微环境阵列的制备

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

Novel Process for 3D Printing Decellularized Matrices

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can provide mechanical stability similar to native tissue while biologic agents offer compositional cues to progenitor cells, leading to their migration, proliferation, and differentiation to reconstitute the original tissues/organs1,2. Unfortunately, many 3D printing compatible, bioresorbable polymers (such as polylactic acid, PLA) are printed at temperatures of 210 &#176;C or higher - temperatures that are detrimental to biologics. On the other hand, polycaprolactone (PCL), a different type of polyester, is a bioresorbable, 3D printable material that has a gentler printing temperature of 65 &#176;C. Therefore, it was hypothesized that decellularized extracellular matrix (DM) contained within a thermally protective PLA barrier could be printed within PCL filament and remain in its functional conformation. In this work, osteochondral repair was the application for which the hypothesis was tested. As such, porcine cartilage was decellularized and encapsulated in polylactic acid (PLA) microspheres which were then extruded with polycaprolactone (PCL) into filament to produce 3D constructs via fused deposition modeling. The constructs with or without the microspheres (PLA-DM/PCL and PCL(-), respectively) were evaluated for differences in surface features.

This protocol describes the production of polycaprolactone (PCL) filament with embedded polylactic acid (PLA) microspheres which contain decellularized matrices (DM) for 3D printing of structural tissue engineering constructs.

一种新的三维打印解光矩阵处理方法

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

趋化梯度多细胞响应的三维分析

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...

Preparation, Administration, and Assessment of In Vivo Tissue-Specific Cellular Uptake of Fluorescent Dye-Labeled Liposomes

There is a growing interest in using liposomes to deliver compounds in vivo particularly for targeted treatment approaches. Depending on the liposome formulation, liposomes may be preferentially taken up by different cell types in the body. This may influence the efficacy of the therapeutic particle as progression of different diseases is tissue- and cell-type-specific. In this protocol, we present one method for synthesizing and fluorescently labeling liposomes using DSPC, cholesterol, and PEG-2000 DSPE and the lipid dye DiD as a fluorescent label. This protocol also presents an approach for delivering liposomes in vivo and assessing cell-specific uptake of liposomes using flow cytometry. This approach can be used to determine the types of cells that take up liposomes and quantify the distribution and proportion of liposome-uptake across cell types and tissues. While not mentioned in this protocol, additional assays such as immunofluorescence and single-cell fluorescence imaging on a cytometer will strengthen any findings or conclusions made as they permit assessment of intracellular staining. Protocols may also need to be adapted depending on the tissue(s) of interest.

The goal of this protocol is to synthesize fluorescently-labeled liposomes and use flow cytometry to identify in vivo localization of liposomes at a cellular level.

荧光染料标记脂质体体内组织特异性细胞摄取的制备、给药和评估

There is a growing interest in using liposomes to deliver compounds in vivo particularly for targeted treatment approaches. Depending on the liposome ...