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1.准备盖玻片的分配数量<ol><li>清洁12毫米盖玻片低棉绒抹布。 </li><li>火焰12毫米盖玻片和14毫米盖玻片在35个毫米的玻璃底菜的微孔，通过使用镊子本生灯火焰传递它们。 </li><li>对待微孔用200μl的0.1N NaOH中进行5分钟，在室温下进行。 </li><li>吸液和空气干燥的微孔30分钟。 </li><li>对待微孔用50-100微升3-氨基丙基三甲为10分钟，在室温下于通风柜。这种化学反应，用胶;因此，使用玻璃吸管，不填微孔完全避免与菜的塑料接触。 </li><li>洗用超纯水微孔约10分钟，直到3-氨基丙基三甲变得清晰。 </li><li>冲洗用的超纯水两次用挤压瓶的微孔。 </li><li>用2毫升的超纯水洗涤微孔吨室温在摇杆定在一个中等速度（约1赫兹）10分钟。 </li><li>吸液和空气干燥的微孔30分钟。 </li><li>对待微孔用2ml 0.5％戊二醛溶液在室温下于一摇臂设置在中等速度（约1赫兹）30分钟。 </li><li>在室温下于一摇臂设置以中速（约1赫兹）10分钟洗涤用2毫升的超纯水的微孔。重复2次以上，总共30分钟。 </li><li>干微孔在一个陡峭的角度（60°或更大）30分钟。 注:盘可以存储2个月在干燥器。 </li></ol> 2.准备的PaaS的侵袭伪足测定<ol><li>为软的PAA（8％丙烯酰胺和0.05％BIS）的1毫升溶液，结合加入200μl40％的丙烯酰胺，25微升的2％的BIS，以及574微升的超纯水（ 表1）。 </li><li>对于硬的PAA（8％丙烯酰胺和0.35％BIS）的1毫升溶液，结合加入200μl40％的丙烯酰胺，175＃的181;升2％的BIS，和409微升的超纯水（ 表1）。 </li><li>为刚性的PAA（12％丙烯酰胺和0.60％BIS）的1毫升溶液，结合300微升40％的丙烯酰胺，300微升2％的BIS，以及169微升的超纯水（ 表1）。 </li><li>脱气15分钟的解决方案。在超纯水中添加200，215，和230微升的1毫克/毫升人血浆纤连蛋白的软，硬，和刚性的PAA溶液，分别。所有的溶液，加入1微升10毫克/毫升丙烯酸N-羟基琥珀酰亚胺（NHS）酯，5微升100毫克/毫升的过硫酸铵，和2微升的N，N，N&#39;，N&#39;-四甲基乙二胺（TEMED ; 表1）。混合轻轻吹打和避免解决方案产生气泡。 </li></ol><table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always"><tbody><tr><td> PAA （1毫升）中</td><td> 40％ AA （微升） </td><td> 2％ （微升） </td><td>超纯 水 （微升） </td><td> 1mg / ml的 FN （微升） </td><td style="padding-left: 4px; padding-right: 4px;"> 10毫克/毫升 NHS酯 （微升） </td><td style="padding-left: 4px; padding-right: 4px;"> 100毫克/毫升 APS （微升） </td><td> TEMED （微升） </td><td>弹 系数 （PA） </td></tr><tr><td>软</td><td> 200 </td><td> 25 </td><td> 574 </td><td> 200 </td><td> 1 </td><td>五</td><td> 2 </td><td> 1023 </td></tr><tr><td>硬</td><td> 200 </td><td> 175 </td><td> 409 </td><td> 215 </td><td> 1 </td><td>五</td><td> 2 </td><td> 7307 </td></tr><tr><td>严格</td><td> 300 </td><td> 300 </td><td> 169 </td><td> 230 </td><td> 1 </td><td>五</td><td> 2 </td><td> 22692 </td></tr></tbody></table> 表1:组分VOLUMES以及由此产生的弹性模量在侵袭伪足实验中使用的软，硬，和严格的分配数量。AA =丙烯酰胺，FN =纤连蛋白，APS =过硫酸铵。 <ol start="5"><li>吸取到每个微孔的中心8.48微升的PAA解决方案。 注意:此卷理论上产生的75微米厚的PAA。 </li><li>轻轻放下上使用镊子微孔液滴的12毫米盖玻片火烧面。 </li><li>允许夹持的PAA溶液聚合15-30分钟。通过检查剩余的解决方案验证聚合。 </li><li>加2ml的1×PBS中，以每个培养皿用镊子取出12毫米盖玻片。 注:10倍PBS中是在实验室和0.011 M KH 2 PO 4，1.54 M氯化钠，和0.056，1M的Na 2 HPO 4组成。 </li><li>用2ml的1×PBS在室温下洗涤​​微孔5分钟。重复2次以上，总共15分钟。 </li></ol> 3. P部分分配的牵引力化验赔偿<ol><li>为软的PAA（8％丙烯酰胺和0.05％BIS）的1毫升溶液，结合加入200μl40％的丙烯酰胺，25微升的2％的BIS，以及566微升的超纯水（ 见表2）。 </li><li>对于硬的PAA（8％丙烯酰胺和0.35％BIS）的1毫升溶液，结合加入200μl40％的丙烯酰胺，175微升2％的BIS，以及401微升的超纯水（ 见表2）。 </li><li>为刚性的PAA（12％丙烯酰胺和0.60％BIS）的1毫升溶液，结合300微升40％的丙烯酰胺，300微升2％的BIS，以及161微升的超纯水（ 见表2）。 </li><li>脱气15分钟的解决方案。添加200，215，和230微升的1毫克/毫升人血浆纤连蛋白的软，硬，和刚性的PAA溶液，分别。声处理8微升200nm的红色荧光微球（的六百零五分之五百八十零nm激发/发射）下30秒。对于每种溶液，加入8微升200nm的红色荧光的微球，1微升10毫克/毫升丙烯酸NHS酯，5微升100毫克/毫升的过硫酸铵，及2微升TEMED（ 见表2）。混合轻轻吹打和避免解决方案产生气泡。 </li></ol><table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always"><tbody><tr><td style="width:40px"> PAA （1毫升）中</td><td> 40％ AA （微升） </td><td> 2％ BIS （微升） </td><td style="padding-left: 4px; padding-right: 4px;">超纯 水 （微升） </td><td> 1mg / ml的 FN （微升） </td><td> 微球 （微升） </td><td style="padding-left: 4px; padding-right: 4px;"> 10毫克/毫升 NHS酯 （微升） </td><td style="padding-left: 4px; padding-right: 4px;"> 100毫克/毫升 APS （微升） </td><td> TEMED （微升） </td><td>弹 系数 （PA） </td></tr><tr><td>软</td><td> 200 </td><td> 25 </td><td> 566 </td><td> 200 </td><td> 8 </td><td> 1 </td><td>五</td><td> 2 </td><td> 1023 </td></tr><tr><td>硬</td><td> 200 </td><td> 175 </td><td> 401 </td><td> 215 </td><td> 8 </td><td> 1 </td><td>五</td><td> 2 </td><td> 7307 </td></tr><tr><td>严格</td><td> 300 </td><td> 300 </td><td> 161 </td><td> 230 </td><td> 8 </td><td> 1 </td><td>五</td><td> 2 </td><td> 22692 </td></tr></tbody></table> 表2:成分量和产生的弹性模量在牵引力试验使用的软，硬，和僵化的分配数量 AA =丙烯酰胺，FN =纤连蛋白，APS =过硫酸铵。 <ol start="5"><li>吸取到每个微孔的中心8.48微升的PAA解决方案。 注意:此卷理论上产生的75微米厚的PAA。 </li><li>轻轻放下日的火烧面Ë12毫米盖玻片上使用镊子微孔液滴。 </li><li>允许夹持的PAA溶液聚合15-30分钟。通过检查剩余的解决方案验证聚合。 </li><li>加2ml的1×PBS中，以每个培养皿用镊子取出12毫米盖玻片。 </li><li>用2ml的1×PBS在室温下洗涤​​微孔5分钟。重复2次以上，总共15分钟。 </li></ol> 4.准备ECM的侵袭伪足测定<ol><li>加热该明胶溶液（1％明胶和1％的蔗糖），以37℃。 </li><li>从微孔的底部保持在玻璃底培养皿以45°角时，治疗用150微升的明胶溶液的部分分配为1分钟，然后小心地吸出。 </li><li>干明胶对部分分配剩余的薄层中的微孔在一个陡峭的角度（60°或更大），以优化干燥60分钟。 </li><li>治疗用2毫升冷却的0.5％戊二醛溶液微孔i上CE为15分钟，随后室温下搅拌30分钟。 </li><li>洗用2毫升的1×PBS的微孔5分钟。重复2次以上，总共15分钟。 </li><li>对待微孔用2ml的1分钟加入硼氢化钠溶液中。轻轻拍打板凳上的菜肴以去除形成明胶的表面上的泡沫。 </li><li>洗板微孔用2毫升1×PBS中5分钟。重复2次以上，总共15分钟。 </li><li>稀释FITC标记的人血浆纤连蛋白溶液用一个1×PBS中50μg/ ml的和离心175000×g离心在4℃下进行15分钟。无论是购买市售的标记纤维连接蛋白或如下标注为: <ol><li>溶解5毫克纤连蛋白在透析管10毫升硼酸盐缓冲液（170毫偏硼酸钠四水合物和40 mM氯化钠）和地点。 </li><li>通过将在200毫升含有6毫克溶解FITC的硼酸盐缓冲液的管在室温下在磁力搅拌器上标记的纤连蛋白设定在1.5小时在黑暗中的最低设置。 </li><li>用500毫升的1×PBS的磁力搅拌器上在黑暗的冷室（4℃）用2体积设定在最低设置3天透析改变了一天。 </li><li> （1:200）的基础上的稀释的等分试样的光密度计算的FITC-标记的纤连蛋白浓度在280纳米和493纳米[OD 280 - （0.36 X OD 493）] / 1.4倍的200次2稀释因子（占为甘油浓缩纤连蛋白中的下一个步骤）所得的以mg / ml单位。 </li><li>在磁力搅拌器上过夜在黑暗的冷室，在4℃下设置在最低设置为50％的甘油溶液透析过夜。） </li></ol></li><li>治疗与150微升，在室温下的FITC标记纤维连接蛋白溶液在黑暗中60分钟的微孔。 </li><li>保持在玻璃底培养皿在时小心吸从微孔底部的FITC-标记的纤连蛋白溶液一个45°角，然后填以70％的乙醇溶液中的玻璃底培养皿在室温下放置10分钟，在黑暗的细胞培养罩进行杀菌。还填充玻璃底培养皿盖或浸泡在70％的乙醇的低掉毛抹布擦拭干净。 </li><li>与1X PBS填补他们5分钟洗玻璃底菜。重复两次用2ml的1×PBS的总共15分钟。 </li></ol> 5.准备侵袭伪足化验和影像<ol><li>添加25000癌细胞在2ml侵袭伪足介质（1:1的DMEM和RPMI 1640，用5％的低蛋白质血清，10％胎牛血清，并新鲜加入表皮生长因子20纳克/毫升），以在玻璃底培养皿和孵育过夜。 </li><li>抽吸和治疗用2ml在室温下在黑暗中的3.7％多聚甲醛溶液的微孔20分钟以固定细胞。 </li><li>后两个快速洗涤用2ml的1×PBS中，治疗的微孔用2ml的0.1％的Triton X-100溶液，在室温坦佩叉涂抹在黑暗中5分钟，以透化细胞。 </li><li>后1快洗用2ml的1×PBS中，治疗用3％牛血清白蛋白阻断在室温下溶液在黑暗中进行60分钟，以阻断细胞的微孔。 </li><li>孵育150微升小鼠抗皮层蛋白抗体（1:750）的微孔阻塞，在室温下在黑暗中的溶液60分钟。 </li><li>用2ml的1×PBS中5分钟洗微孔。重复2次以上，总共15分钟。 </li><li>孵育微孔用150微升的山羊抗小鼠633抗体（1:500）和546鬼笔环肽（1:750），在封闭，在室温下在黑暗中的溶液60分钟。 </li><li>用2ml的1×PBS中5分钟洗微孔。重复2次以上，总共15分钟。 </li><li>加入6滴封固剂来填充每个微孔，盖上22×22盖玻片。 </li><li>图像皮层蛋白和肌动蛋白使用647分之632nm激发和发射滤光片，以确定侵袭伪足和五百七十〇分之五百五十六纳米，分别使用高NA，40X油浸镜头上的宽视场倒置显微镜。同样地，图像纤连蛋白使用的五百一十八分之四百九十二纳米的激发和发射滤光器识别ECM降解。 </li></ol> 6.制备及牵引力化验成像<ol><li>在2毫升侵袭伪足中添加15000癌细胞玻璃底菜，并培育过夜。 </li><li>用2ml的L-15培养基中，在玻璃底培养皿抽吸培养基并更换，具有相同的补充剂（5％低蛋白血清，10％胎牛血清，并新鲜加入表皮生长因子20纳克/毫升），预-warmed至37℃。 </li><li>孵育在倒置显微镜预平衡至37℃的高湿度下1小时，在玻璃底培养皿中的环境室中。 </li><li>就拿四套使用高NA，40X镜头上的宽视场倒置显微镜自动化阶段，以纪念蜂窝位置的图像: <ol><li>对图像细胞P使用相衬（"阶段"的形象）将PAAS的。 </li><li>使用六百四十五分之五百六十〇nm激发和发射滤光片由下将细胞略微聚焦直到微球在部分分配的顶部的第一层图像的荧光微球进入对焦（"紧张"的图像）。 </li><li>此外，集中于部分分配（使用微球体作为标记）的底部，并拍摄图像（"底部"的图像）。注意:此图像的目的是记录的z位置，如代表结果说明得到的实际的PAA厚度在每个位置上用于牵引力的计算。 </li><li>之后，这些图像已被收购的多个位置，轻轻在220微升的10％的Triton-X解决方案，混合，除去细胞。在部分分配的顶部在每个位置上如先前所述（"空"的图像）的图像的微球。 </li></ol></li></ol>

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	<li>Paszek, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensional+homeostasis+and+the+malignant+phenotype.">Tensional homeostasis and the malignant phenotype.</a> Cancer cell. 8, 241-254 (2005).</li><li>Jaalouk, D. E., Lammerding, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanotransduction+gone+awry.">Mechanotransduction gone awry.</a> Nature. 10, 63-73 (2009).</li><li>Paszek, M. J., Weaver, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tension+mounts:+mechanics+meets+morphogenesis+and+malignancy.">The tension mounts: mechanics meets morphogenesis and malignancy.</a> Journal of mammary gland biology and neoplasia. 9, 325-342 (2004).</li><li>Parekh, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensing+and+modulation+of+invadopodia+across+a+wide+range+of+rigidities.">Sensing and modulation of invadopodia across a wide range of rigidities.</a> Biophysical. 100, 573-582 (2011).</li><li>Jerrell, R. J., Parekh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+traction+stresses+mediate+extracellular+matrix+degradation+by+invadopodia.">Cellular traction stresses mediate extracellular matrix degradation by invadopodia.</a> Acta biomaterialia. 10, 1886-1896 (2014).</li><li>Kraning-Rush, C. M., Califano, J. P., Reinhart-King, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+traction+stresses+increase+with+increasing+metastatic+potential.">Cellular traction stresses increase with increasing metastatic potential.</a> PLoS ONE. 7, e32572 (2012).</li><li>Haage, A., Nam, D. H., Ge, X., Schneider, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinase-14+is+a+mechanically+regulated+activator+of+secreted+MMPs+and+invasion.">Matrix metalloproteinase-14 is a mechanically regulated activator of secreted MMPs and invasion.</a> Biochemical and biophysical research communications. , (2014).</li><li>Haage, A., Schneider, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+contractility+and+extracellular+matrix+stiffness+regulate+matrix+metalloproteinase+activity+in+pancreatic+cancer+cells.">Cellular contractility and extracellular matrix stiffness regulate matrix metalloproteinase activity in pancreatic cancer cells.</a> FASEB J. , (2014).</li><li>Weaver, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invadopodia:+specialized+cell+structures+for+cancer+invasion.">Invadopodia: specialized cell structures for cancer invasion.</a> Clinical &amp; experimental metastasis. 23, 97-105 (2006).</li><li>Weaver, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invadopodia.">Invadopodia.</a> Curr Biol. 18, R362-364 (2008).</li><li>Bravo-Cordero, J. J., Hodgson, L., Condeelis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+cell+invasion+and+migration+during+metastasis.">Directed cell invasion and migration during metastasis.</a> Current opinion in cell biology. 24, 277-283 (2012).</li><li>Alexander, N. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+rigidity+promotes+invadopodia+activity.">Extracellular matrix rigidity promotes invadopodia activity.</a> Curr Biol. 18, 1295-1299 (2008).</li><li>Barlow, W. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+breast+cancer+risk+prediction+model+for+women+undergoing+screening+mammography.">Prospective breast cancer risk prediction model for women undergoing screening mammography.</a> Journal of the National Cancer Institute. 98, 1204-1214 (2006).</li><li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projecting+absolute+invasive+breast+cancer+risk+in+white+women+with+a+model+that+includes+mammographic+density.">Projecting absolute invasive breast cancer risk in white women with a model that includes mammographic density.</a> Journal of the National Cancer Institute. 98, 1215-1226 (2006).</li><li>Butcher, D. T., Alliston, T., Weaver, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tense+situation:+forcing+tumour+progression.">A tense situation: forcing tumour progression.</a> Nature reviews. Cancer. 9, 108-122 (2009).</li><li>Zaman, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+of+tumor+cells+in+3D+matrices+is+governed+by+matrix+stiffness+along+with+cell-matrix+adhesion+and+proteolysis.">Migration of tumor cells in 3D matrices is governed by matrix stiffness along with cell-matrix adhesion and proteolysis.</a> Proceedings of the National Academy of Sciences of the United States of America. 103, 10889-10894 (2006).</li><li>Provenzano, P. P., Inman, D. R., Eliceiri, K. W., Keely, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+density-induced+mechanoregulation+of+breast+cell+phenotype,+signaling+and+gene+expression+through+a+FAK-ERK+linkage.">Matrix density-induced mechanoregulation of breast cell phenotype, signaling and gene expression through a FAK-ERK linkage.</a> Oncogene. 28, 4326-4343 (2009).</li><li>Munevar, S., Wang, Y., Dembo, 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+force+microscopy+of+migrating+normal+and+H-ras+transformed+3T3+fibroblasts.">Traction force microscopy of migrating normal and H-ras transformed 3T3 fibroblasts.</a> Biophysical journal. 80, 1744-1757 (2001).</li><li>Rosel, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Up-regulation+of+Rho/ROCK+signaling+in+sarcoma+cells+drives+invasion+and+increased+generation+of+protrusive+forces.">Up-regulation of Rho/ROCK signaling in sarcoma cells drives invasion and increased generation of protrusive forces.</a> Mol Cancer Res. 6, 1410-1420 (2008).</li><li>Indra, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+correlation+of+mechanical+forces+and+metastatic+capacity.">An in vitro correlation of mechanical forces and metastatic capacity.</a> Phys Biol. 8, 015015 (2011).</li><li>Parekh, A., Weaver, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cancer+invasiveness+by+the+physical+extracellular+matrix+environment.">Regulation of cancer invasiveness by the physical extracellular matrix environment.</a> Cell adhesion &amp; migration. 3, 288-292 (2009).</li><li>Weaver, A. M., Page, J. M., Guelcher, S. A., Parekh, A., Coutts, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Molecular Biology in Adhesion Protein Protocols. 1046, 171-189 (2013).</li><li>Samani, A., Zubovits, J., Plewes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastic+moduli+of+normal+and+pathological+human+breast+tissues:+an+inversion-technique-based+investigation+of+169+samples).">Elastic moduli of normal and pathological human breast tissues: an inversion-technique-based investigation of 169 samples).</a> Physics in medicine and biology. 52, 1565-1576 (2007).</li><li>Wang, J. H., Lin, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+traction+force+and+measurement+methods.">Cell traction force and measurement methods.</a> Biomechanics and modeling in mechanobiology. 6, 361-371 (2007).</li><li>Dembo, M., Oliver, T., Ishihara, A., 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=Imaging+the+traction+stresses+exerted+by+locomoting+cells+with+the+elastic+substratum+method.">Imaging the traction stresses exerted by locomoting cells with the elastic substratum method.</a> Biophysical journal. 70, 2008-2022 (1996).</li><li>Dembo, M., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stresses+at+the+cell-to-substrate+interface+during+locomotion+of+fibroblasts.">Stresses at the cell-to-substrate interface during locomotion of fibroblasts.</a> Biophysical journal. 76, 2307-2316 (1999).</li><li>Engler, A. J., Rehfeldt, F., Sen, S., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtissue+elasticity:+measurements+by+atomic+force+microscopy+and+its+influence+on+cell+differentiation.">Microtissue elasticity: measurements by atomic force microscopy and its influence on cell differentiation.</a> Methods Cell Biol. 83, 521-545 (2007).</li><li>Kandow, C. E., Georges, P. C., Janmey, P. A., Beningo, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyacrylamide+hydrogels+for+cell+mechanics:+steps+toward+optimization+and+alternative+uses.">Polyacrylamide hydrogels for cell mechanics: steps toward optimization and alternative uses.</a> Methods in cell biology. 83, 29-46 (2007).</li><li>Leach, J. B., Brown, X. Q., Jacot, J. G., Dimilla, P. A., 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=Neurite+outgrowth+and+branching+of+PC12+cells+on+very+soft+substrates+sharply+decreases+below+a+threshold+of+substrate+rigidity.">Neurite outgrowth and branching of PC12 cells on very soft substrates sharply decreases below a threshold of substrate rigidity.</a> J Neural Eng. 4, 26-34 (2007).</li><li>Zhou, J., Kim, H. Y., Wang, J. H., Davidson, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macroscopic+stiffening+of+embryonic+tissues+via+microtubules,+RhoGEF+and+the+assembly+of+contractile+bundles+of+actomyosin.">Macroscopic stiffening of embryonic tissues via microtubules, RhoGEF and the assembly of contractile bundles of actomyosin.</a> Development. 137, 2785-2794 (2010).</li><li>Buxboim, A., Rajagopal, K., Brown, A. E., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+deeply+cells+feel:+methods+for+thin+gels.">How deeply cells feel: methods for thin gels.</a> J Phys Condens Matter. 22, 194116 (2010).</li></ol>

这项工作是由美国国立卫生研究院在奖号K25CA143412美国国家癌症研究所（帕雷克）的支持。我们要感谢额外的支持是由耳鼻咽喉科提供。内容完全是作者的责任，并不一定代表美国国立卫生研究院的官方意见。

我们提出的用于制造部分分配，可以用来作为基础侵袭伪足和牵引力测定法来关联侵入性和收缩的细胞行为的方法。虽然部分分配额早已看惯了对细胞的刚性作用，并计算牵引力18,24,27，该协议是第一个开发基于分配数量与同一刚性平行检测，以响应矩阵相关侵入性和收缩的细胞行为机械性能。正确激活的玻璃底菜盖玻片确保分配数量将绑定到他们，不会脱落。虽然我们和其他人已经依靠...

肿瘤相关的ECM的刚性已被确定为在通过增加肌动球蛋白收缩1-3找到恶性行为一显著因素。虽然这种效应已主要被证明与乳腺癌细胞，基质刚性已经发现改变从多种癌症4-8表明肿瘤刚度可以以其它类型的癌症中发挥作用的来源的细胞侵入性。渗透侵入迁移过程中，交联组织，癌细胞利用肌动蛋白丰富的被称为侵袭伪足的本地化蛋白酶来灶性降低ECM 9粘合突起。侵袭伪足被认为是侵入细胞的标志，并有牵连的肿瘤细胞浸润和转移10,11。以前的工作已经表明，基质刚度可以调节侵袭伪足数目和通过肌球蛋白II活性和机械敏感蛋白12相关的ECM降解4,12。鉴于肿瘤密度和癌症侵袭性13,14之间的相关性，这些结果表明通过该癌细胞可能响应刚性肿瘤组织通过肌动球蛋白收缩驱动侵袭和转移的机制。 在体外 ECM刚性和体内组织密度已显示调节癌细胞1,15-17侵袭行为。而肌动球蛋白收缩似乎是重要的在此过程中，目前的研究冲突，以转移能力是否相关，增加或减少收缩力6,18-20。此外，目前还不清楚是否这些力量直接介导侵袭伪足的活动21。我们最近发现，肿瘤细胞收缩力是依赖于矩阵的刚度和被预测ECM降解受侵袭伪足5。这些结果表明，蜂窝力可能通过介导侵袭伪足的交流在癌症发展中起重要作用tivity响应于肿瘤微环境的机械性能。 为了关联癌细胞5侵袭和收缩性能，我们修改了协议，用于创建分配数量与以前用于调查的刚性依赖的侵袭伪足的活动4,12,22不同的僵化。通过化学整个部分分配交联的人血浆纤连蛋白，这些修改后的水凝胶可以用作基础为侵袭伪足和牵引力试验，以确保细胞经历了同样的刚性在两个实验5。在侵袭伪足测定中，纤连蛋白提供了天然的结合域为明胶以重叠的ECM链接到部分分配给检测基质降解。在牵引力测定中，纤连蛋白提供的配体的直接的细胞粘连，以检测用来计算蜂窝牵引力微球的位移。这种方法会导致我们所谓的软，硬，和僵化的分配数量绑定到玻璃底菜，并有弹性模量，E，1023，7307和22692帕5的跨度报道了正常和癌变组织23机械性能的范围内。

<table><tbody><tr><td>3-Aminopropyltrimethoxysilane</td><td>Sigma-Aldrich</td><td>281778</td><td></td></tr><tr><td>Acrylamide (40%)</td><td>Bio-Rad</td><td>161-0140</td><td></td></tr><tr><td>Acrylic acid NHS ester</td><td>Sigma-Aldrich</td><td>A8060</td><td>prepare fresh in fume hood 10 mg/ml in DMSO</td></tr><tr><td>Alexa Fluor 546 phalloidin</td><td>Life Technologies</td><td>A22283</td><td>can also use rhodamine</td></tr><tr><td>Ammonium persulfate</td><td>Bio-Rad</td><td>161-0700</td><td>prepare fresh 10% solution in 1x PBS</td></tr><tr><td>Aqua Poly/Mount</td><td>Polysciences</td><td>18606</td><td>use 6 drops to fill microwells</td></tr><tr><td>BIS (2%)</td><td>Bio-Rad</td><td>161-0142</td><td></td></tr><tr><td>Bovine serum albumin</td><td>RPI</td><td>A30075</td><td>make 3% for blocking solution in 1x PBS and store in 4 &amp;deg;C</td></tr><tr><td>Coverslips (12 mm)</td><td>Fisher Scientific</td><td>12-545-80</td><td></td></tr><tr><td>dialysis tubing</td><td>Sigma-Aldrich</td><td>D9777</td><td>pre-equilibrate in borate buffer for 15 - 30 min</td></tr><tr><td>DMEM</td><td>Cellgro</td><td>10-013-CV</td><td>use to make invadopodia medium</td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D8418</td><td>use to make acrylic acid NHS ester solution</td></tr><tr><td>Epidermal growth factor</td><td>Life Technologies</td><td>PHG0311</td><td>use to make invadopodia medium</td></tr><tr><td>Ethanol</td><td>PHARMCO-AAPER</td><td>E200</td><td>dilute with ultrapure water to 70%</td></tr><tr><td>FBS</td><td>Thermo Scientific</td><td>SH30070.03</td><td>use to make invadopodia medium</td></tr><tr><td>FITC</td><td>Sigma-Aldrich</td><td>F7250</td><td>protect from light</td></tr><tr><td>Gelatin</td><td>Polysciences</td><td>00639</td><td>typically make 10 ml of 1% sucrose/1% gelatin solution in PBS and store at 4 &amp;deg;C (preheat PBS to dissolve gelatin easily)</td></tr><tr><td>Glass bottom dishes (35 mm coverslips)</td><td>MatTek</td><td>P35G-0-14-C</td><td>coverslips are uncoated</td></tr><tr><td>Glutaraldehyde (25%)</td><td>Polysciences</td><td>01909</td><td>dilute with 1x PBS to 0.5%</td></tr><tr><td>Goat anti-mouse Alexa Fluor 633 antibody</td><td>Life Technologies</td><td>A21050</td><td></td></tr><tr><td>Human plasma fibronectin</td><td>Life Technologies</td><td>33016-015</td><td>add 5 ml of ultrapure water to make 1 mg/ml; aliquot in volumes based on use to avoid excessive freezing and thawing cycles</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>EMD Millipore</td><td>PX-1565-1</td><td>use to make 10x PBS stock</td></tr><tr><td>Mouse anti-cortactin 4F11 antibody</td><td>EMD Millipore</td><td>05-180</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>EMD Millipore</td><td>SX-0720-1</td><td>use to make 10x PBS stock</td></tr><tr><td>NaCl</td><td>RPI</td><td>S23020</td><td>use to make 10x PBS stock and borate buffer</td></tr><tr><td>NaOH (1 N)</td><td>Sigma-Aldrich</td><td>S2770</td><td>dilute with ultrapure water to 0.1 N</td></tr><tr><td>Nu-Serum (low-protein serum)</td><td>BD Biosciences</td><td>355500</td><td>use to make invadopodia medium</td></tr><tr><td>Paraformaldehyde</td><td>Acros</td><td>416785000</td><td>typically make 10% stock in 1x PBS, prepare in fume hood, and add a few ml of strong NaOH to dissolve paraformaldehyde easily then bring back to pH 7.4 with strong HCl)</td></tr><tr><td>PBS (sterile)</td><td>Cellgro</td><td>21-040-CV</td><td>use for cell culture</td></tr><tr><td>RPMI 1640</td><td>Cellgro</td><td>10-040-CV</td><td>use to make invadopodia medium</td></tr><tr><td>Sodium borohydride</td><td>Sigma-Aldrich</td><td>452882</td><td>prepare fresh in fume hood 1 mg/ml in 1x PBS</td></tr><tr><td>Sodium metaborate tetrahydrate</td><td>Sigma-Aldrich</td><td>S0251</td><td>use to make borate buffer</td></tr><tr><td>Sucrose</td><td>RPI</td><td>S24060</td><td>typically make 10 ml of 1% sucrose/1% gelatin solution in PBS and store at 4 &amp;deg;C (preheat PBS to dissolve gelatin easily)</td></tr><tr><td>TEMED</td><td>Bio-Rad</td><td>161-0800</td><td></td></tr><tr><td>Triton X-100</td><td>Alfa Aesar</td><td>A16046</td><td>make 10% stock in 1x PBS and use as is for cell removal in traction force assay or dilute with 1x PBS for staining</td></tr></tbody></table>

polyacrylamide gels for invadopodia and traction force assays on cancer cells

刚性肿瘤组织中一直强烈牵连在调节癌细胞的迁移和侵袭。通过交联组织侵袭性迁移是由肌动蛋白富突起称为侵袭伪足该蛋白水解降解细胞外基质（ECM）提供便利。侵袭伪足的活动已被证明是依赖于ECM的刚性和癌细胞收缩力表明刚性信号可以通过肌动球蛋白收缩调节这些亚细胞结构。癌细胞的侵袭性和收缩性能可在体外使用基于不同的刚性聚丙烯酰胺凝胶（分配数量）侵袭伪足和牵引力试验相关。癌细胞的侵袭性和收缩性能可在体外使用基于不同的刚性聚丙烯酰胺凝胶（分配数量）侵袭伪足和牵引力试验相关。而在两个测定之间一些变型存在，这里介绍的协议提供了用于第创建部分分配的方法在可以在两个测定中使用，并且很容易适应用户的特定生物和技术的需求。

在侵袭伪足测定中，侵袭伪足通常标识标记像肌动蛋白和皮层蛋白在细胞体中的点状结构（ 图1）的共定位。既积极降解和非恶化侵袭伪足可以计数和由这些结构是否共定位与缺乏的FITC标记的纤连蛋白（ 图1）的荧光信号的黑色区域是有区别的。侵袭伪足手动计数，而每个单元ECM降解由细胞的轮廓内手动阈值处理这些黑色区域来确定。 在牵引力测定?...

Watch this Scientific Journal Video about Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells at JoVE.com

Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells

在肿瘤微机械刚性起着通过增加侵袭伪足活性和肌动球蛋白收缩驱动恶性行为了至关重要的作用。用聚丙烯酰胺凝胶（部分分配），侵袭伪足和牵引力测定可以被用于研究癌细胞的侵入性和收缩特性响应于矩阵刚性。

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 ...

polyacrylamide-gels-for-invadopodia-traction-force-assays-on-cancer

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

12.0K 次观看.  Vanderbilt University Medical Center. 在肿瘤微机械刚性起着通过增加侵袭伪足活性和肌动球蛋白收缩驱动恶性行为了至关重要的作用。用聚丙烯酰胺凝胶（部分分配），侵袭伪足和牵引力测定可以被用于研究癌细胞的侵入性和收缩特性响应于矩阵刚性。 

视频: Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells

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

Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their extracellular matrix (ECM) environment. Eukaryotic cells constantly sense their local microenvironment through surface mechanosensors to transduce physical changes of ECM into biochemical signals, and integrate these signals to achieve specific changes in gene expression. Interestingly, physicochemical and mechanical parameters of the ECM can couple with each other to regulate cell fate. Therefore, a key to understanding mechanotransduction is to decouple the relative contribution of ECM cues on cellular functions.
Here we present a detailed experimental protocol to rapidly and easily generate biologically relevant hydrogels for the independent tuning of mechanotransduction cues in vitro. We chemically modified polyacrylamide hydrogels (PAAm) to surmount their intrinsically non-adhesive properties by incorporating hydroxyl-functionalized acrylamide monomers during the polymerization. We obtained a novel PAAm hydrogel, called hydroxy-PAAm, which permits immobilization of any desired nature of ECM proteins. The combination of hydroxy-PAAm hydrogels with microcontact printing allows to independently control the morphology of single-cells, the matrix stiffness, the nature and the density of ECM proteins. We provide a simple and rapid method that can be set up in every biology lab to study in vitro cell mechanotransduction processes. We validate this novel two-dimensional platform by conducting experiments on endothelial cells that demonstrate a mechanical coupling between ECM stiffness and the nucleus.

We present a new polyacrylamide hydrogel, called hydroxy-PAAm, that allows a direct binding of ECM proteins with minimal cost or expertise. The combination of hydroxy-PAAm hydrogels with microcontact printing facilitates independent control of many cues of the natural cell microenvironment for studying cellular mechanostransduction.

去耦力学传递线索的影响制备羟基聚丙烯酰胺水凝胶的

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their ...

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

从手术组织小学的人结肠癌细胞的分离和培养直接它们柔软的弹性基底牵引术

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

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.

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 ...

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

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts 

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

定量测量的侵袭伪足介导的细胞外基质蛋白水解单和多细胞的情况下

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

生物学

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 ...

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, the effect of tissue elasticity on cell function is a major area of mechanobiology research because tissue stiffness modulates with disease, development, and injury. Static tissue-mimicking materials, or materials that cannot alter stiffness once cells are plated, are predominately used to investigate the effects of tissue stiffness on cell functions. While information gathered from static studies is valuable, these studies are not indicative of the dynamic nature of the cellular microenvironment in vivo. To better address the effects of dynamic stiffness on cell function, we developed a DNA-crosslinked polyacrylamide hydrogel system (DNA gels). Unlike other dynamic substrates, DNA gels have the ability to decrease or increase in stiffness after fabrication without stimuli. DNA gels consist of DNA crosslinks that are polymerized into a polyacrylamide backbone. Adding and removing crosslinks via delivery of single-stranded DNA allows temporal, spatial, and reversible control of gel elasticity. We have shown in previous reports that dynamic modulation of DNA gel elasticity influences fibroblast and neuron behavior. In this report and video, we provide a schematic that describes the DNA gel crosslinking mechanisms and step-by-step instructions on the preparation DNA gels.

Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.

DNA交联聚丙烯酰胺水凝胶的制备

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, ...

Analyses of Actin Dynamics, Clutch Coupling and Traction Force for Growth Cone Advance

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes depend on the advances of growth cones that located at the tips of neurites. Axonal growth cones generate driving forces by sensing their local microenvironment and modulating cytoskeletal dynamics and actin-adhesion coupling (clutch coupling). Decades of research have led to the identification of guidance molecules, their receptors, and downstream signaling cascades for regulating neuronal migration and axonal guidance; however, the molecular machineries required for generating forces to drive growth cone advance and navigation are just beginning to be elucidated. At the leading edge of neuronal growth cones, actin filaments undergo retrograde flow, which is powered by actin polymerization and actomyosin contraction. A clutch coupling between F-actin retrograde flow and adhesive substrate generates traction forces for growth cone advance. The present study describes a detailed protocol for monitoring F-actin retrograde flow by single speckle imaging. Importantly, when combined with an F-actin marker Lifeact, this technique can quantify 1) the F-actin polymerization rate and 2) the clutch coupling efficiency between F-actin retrograde flow and the adhesive substrate. Both are critical variables for generating forces for growth cone advance and navigation. In addition, the present study describes a detailed protocol of traction force microscopy, which can quantify 3) traction force generated by growth cones. Thus, by coupling the analyses of&#160;single speckle imaging and traction force microscopy, investigators can monitor the molecular mechanics underlying growth cone advance and navigation.

To advance, growth cones must exert traction forces against the external environment. The generation of traction forces is dependent on actin dynamics and clutch coupling. The present study describes methods for analyzing actin dynamics, clutch coupling and traction forces for growth cone advance.

肌动蛋白动力学、离合器耦合和牵引力分析，促进生长锥体前进

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes ...