Name: using cell-substrate impedance and live cell imaging to measure real-time changes in cellular adhesion and de-adhesion induced by matrix modification
Uploaded: 2015-02-19T13:00:00.000+00:00
Duration: 9 min 11 s
Description: Watch this Scientific Journal Video about Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification at JoVE.

This work was funded by a National Health and Medical Research Council (NHMRC) Project Grant 568721 (SRT) and, in part, by a UNSW Faculty of Medicine Faculty Research Grant (MDR).

1.一般内皮细胞培养<ol><li>培养牛主动脉内皮细胞在明胶包被的组织培养烧瓶（通道4-9）（涂层的组织培养表面用0.1％w / v的明胶的PBS在RT下15分钟）在EGM-2培养基（用EGM-2子弹试剂盒含有5％胎牛血清，生长因子和由制造商提供的，除了氢化可的松所有补充剂）。 </li><li>当细胞接近铺满（约3天后接种后1:4的分流），收获细胞，通过用0.05％w / v的胰蛋白酶/ 0.02％w / v的EDTA的PBS在37℃。后大部分细胞已经分离，添加完全EGM-2介质以猝灭胰蛋白酶，然后离心（100×g离心5分钟）。 </li><li>制备细胞在内皮细胞上的从头粘附于纤连蛋白的研究（实验1:第2部分）和血管内皮细胞与在该基板上建立的密合性（实验2:第3节）随后脱的附着力。 <ol><li>洗收获细胞一次，用无血清培养基199含有1％w / v的牛血清白蛋白（BSA），并重新离心（100×g离心5分钟）。 </li><li>重悬的细胞在无血清培养基199含有1％w / v的BSA的2.5×10 5个细胞/ ml（细胞-基底阻抗测量）或5×10 5个细胞/ ml（活细胞成像分析），并在37保持℃，在使用前。 注:细胞的粘附反应是温度差高度敏感（ 例如，由于对流的效果）因此，所有的设备和解决方案中使用时下列协议来处理和治疗细胞应保持在37℃的恒定温度下进行。 </li></ol></li></ol> 2.实验1:在本地和MPO氧化纤维连接蛋白（细胞衬底阻抗）量化从头内皮细胞黏附注:实验1探讨在何种程度上MPO介导的纤维连接蛋白的氧化损害其支持从头能力</em&gt;悬浮内皮细胞的粘附。 <ol><li>涂层纤维连接蛋白上的96孔金细胞 - 基底阻抗微电极阵列。添加80微升/孔的纯化牛纤连蛋白在5微克/毫升在PBS中，孵育2小时，在37°C和除去该溶液。 </li><li>孵育纤连蛋白涂覆的表面与MPO允许MPO的结合表面结合的纤连蛋白。添加80微升/孔的纯化的人嗜中性粒细胞的MPO在20纳米中Hank氏平衡盐溶液（HBSS）中并孵育0.5小时，在37℃。 </li><li>表面洗两次HBSS除去未结合的MPO。 </li><li>添加H 2 O 2（0-10μM终浓度）至含有80微升/孔的HBSS以引发微电极阵列板的孔中的MPO催化的，次氯酸依赖性纤连蛋白氧化和孵育另0.5小时，在37℃。 </li><li>审查有关抑制剂或MPO催化的反应的调节剂（ 例如 ，可替代的MPO酶的作用底物，酶抑制剂或抗氧化剂;见9为详细说明），这些现有立即于H 2 O 2的加成添加到HBSS中。 </li><li>经过0.5小时，待表面与蛋氨酸淬火残余的表面结合氧化物质， 也就是说 ，无蛋白结合氯胺，这可能产生混淆的细胞活动。添加10mM甲硫氨酸在80微升HBSS中每孔并孵育10分钟，在37℃。 </li><li>座面与BSA。添加80微升/孔的BSA的0.2％w / v的在PBS中，孵育2小时，在37℃，并除去溶液。 注:残留未涂覆的表面区域会支持细胞粘附和用非黏着蛋白（ 即 ，BSA）阻断这些确保细胞粘附反应严格依赖于所用的纯化的细胞粘接性基质，在这种情况下，纤连蛋白。 </li><li>洗具HBSS表面两次。 注:没有前面的表面处理明显影响细胞基质readinGS。 </li><li>种子悬浮内皮细胞（添加200μl/孔在约2.5×10 5个细胞/ ml，在无血清的培养基199含有1％BSA制备;参见1.3）上的天然或MPO氧化纤连蛋白涂覆的表面。 </li><li>立即种子细胞（ 即 ，之前的任何细胞附着和扩散）后，装入微电极阵列板放置在培养箱口（收纳在37℃的培养箱中5％CO 2的存在下）。 <ol><li>使用仪器的软件立即采取一个"空白"读数正常化随后的细胞 - 基底阻抗（&#39;单元索引&#39;）的值，以在没有细胞粘附中得到的初始背景值。 </li><li>发起收购连续细胞指数数据（至少一个细胞指数读数/分钟）。 </li></ol></li><li>孵育细胞在37℃和5％CO 2下2小时的时间段期间，最大细胞附着和铺展实现;这是由反射细胞指数值的趋于稳定（ 见图3A）。 注:前面的实验（实验1）研究如何纤连蛋白的最初的​​MPO介导的氧化限制了内皮细胞，建立的细胞粘附在该基板上的能力。下面的实验（实验2）研究如何MPO介导的纤连蛋白的氧化促进了细胞-基质粘附性（ 即 ，解密合性）在内皮细胞中既定的附着力减小到该基板。在这两个实验中的治疗方法是基本相同的，除了对MPO介导的纤连蛋白氧化的定时（ 即 ，细胞粘附前-实验1;细胞粘附后-实验2）。 </li></ol> 3.实验2:量化内皮细胞去黏附纤连蛋白，从在应对MPO介导的纤维连接蛋白氧化（细胞衬底阻抗和活细胞成像） <ol><li>涂层纤维连接蛋白上的96孔金微电极一rrays的细胞基质阻抗测量（添加80微升/孔纤维连接蛋白在PBS 5微克/毫升，并培育2小时，在37°C）或35毫米的玻璃底细胞培养皿活细胞成像分析（加2ml /纤连蛋白的皿在PBS中5微克/毫升孵育2小时，在37℃）。 </li><li>座面与BSA。添加BSA的0.2％w / v的在PBS中在3.1所示的卷和孵育2小时，在37℃。 </li><li>孵育MPO表面，使MPO到纤连蛋白的结合。添加20nM的纯化的人MPO在HBSS在3.1所示的卷和孵育0.5小时，在37℃。 </li><li>表面洗两次HBSS除去未结合的MPO。 </li><li>种子悬浮内皮细胞（在无血清培养基199含有1％w / v的BSA的制备;见1.3）上的天然或MPO承载纤连蛋白涂覆的表面。 <ol><li>添加200μl/孔以2.5×10 5个细胞/ ml至96孔细胞-基底阻抗微电极阵列。 </li> &lt;LI&gt;加入2毫升/皿以5×10 5个细胞/ ml至35毫米的玻璃有底细胞培养皿。 </li></ol></li><li>立即种子细胞（ 即之前的任何细胞附着和传播）后: <ol><li>摩的96孔微电极阵列板到细胞-基底阻抗培养箱口（收纳在37℃的培养箱中5％CO 2的存在下），并采取"空白"读数以引发连续采集单元索引数据（参照第2.10节）。 </li><li>转印35毫米玻璃有底细胞培养皿以37℃培养箱中5％CO 2的存在。 </li></ol></li><li>孵育细胞在37℃下2小时，以允许最大的细胞附着和扩散（参照第2.11节）。 </li><li>暂时取出96孔微电极阵列板（暂停细胞指数读数在这一点上）或35毫米的玻璃有底从37℃培养箱细胞培养皿，除去细胞上清液，加入温热（37℃）的HBSS（体积为p呃步3.1）。 注:HBSS采用在研究MPO催化氧化反应，而不是完整的文化传媒，因为后者包含了与氧化反应干扰可氧化的物种。 </li><li>审查抑制剂的效果/ MPO催化的反应（替代酶底物，酶抑制剂或抗氧化剂）或细胞信号抑制剂的调节剂（ 例如 ，40微米的II型肌球蛋白抑制肌动球蛋白的收缩），现在添加这些。 </li><li>立即将微电极阵列板放回37℃培养箱端口（重新开工细胞指数读数在这一点），或者35毫米的玻璃有底细胞培养皿放回37℃培养箱，并且使细胞在平衡HBSS 0.5小时。 注:经过HBSS 0.5小时平衡，细胞指数值稳定在稍低值;当预孵化细胞酶底物，酶和细胞信号传导抑制剂或抗氧化剂，首先确保吨HESE不显著影响平衡后获得的值。 </li><li>发起的MPO催化的，次氯酸依赖性纤连蛋白氧化和脱密合性。 <ol><li>对于使用96孔的微电极阵列板电池阻抗的研究: <ol><li>从培养箱中取出微电极阵列板和暂停细胞指数的测量。 </li><li>添加H 2 O 2（0-10μM终浓度）的HBSS中，并通过重复的移液轻轻混合。 </li><li>随即，重新安装微电极阵列放回37℃培养箱端口，并重新展开收购细胞指数读数。 </li></ol></li><li>对于使用35毫米玻璃见底细胞培养皿活细胞成像的研究: <ol><li>从培养箱中取出培养皿，并安装到一个倒置的共聚焦显微镜装有63X水物镜与合适的DIC的光学记录活细胞的电影的37℃的加热阶段。 </li><li>专注于细胞优化DIC光学（科勒照明，偏迟缓和相机偏置/增益;有关详细信息，请参阅第13）。 </li><li>发起DIC电影和1分钟记录未处理的细胞的基线读数。 </li><li>添加H 2 O 2（0-10μM终浓度），并通过重复的移液轻轻混合。重新聚焦显微镜（如有必要），然后继续处理的细胞的记录DIC电影对于所需的时间段。 </li></ol></li></ol></li></ol> 4.数据分析和介绍<ol><li>细胞基质阻抗数据<ol><li>导出原始数据（细胞指数对时间）到电子表格中。 </li><li>用于细胞去-粘附研究（实验2:第3节），通过在一个值设置为的MPO介导的纤连蛋白的氧化开始记录之前立即值正常化的数据1（ 即 ，紧接在3.11加入的H 2 O 2的前.1.1）。 注意:这可确保相对变化由MPO介导的纤维结合素的氧化引起的细胞指数值不受在井之间的绝对细胞指数值小的初始差别所掩盖。 <ol><li>本数据作为绘图归细胞指数（Y轴）对时间（x轴）的。 </li></ol></li></ol></li><li>活细胞成像数据（细胞去黏附的研究实验2:第3节） <ol><li>打开DIC住前夕和MPO介导的纤维连接蛋白氧化标准的图像分析程序（ 例如，ImageJ的软件）开始后记录细胞成像电影。 </li><li>在至少两个分开的DIC电影随机选择多个细胞和通过手动跟踪它们的膜边缘和量化包围的象素数测量在连续的帧（ 例如，在1分钟的间隔）的投影面积。 </li><li>通过设定值导出的原始数据（投影单元面积对时间）到Excel电子表格和正常化小区区域的数据记录之前立即的MPO介导的纤连蛋白的氧化在值1的启动（ 即 ，紧接在3.11.2.3加入的H 2 O 2之前）。 </li><li>本数据作为标准化的小区区域（Y轴）对时间（x轴）的曲线图。 </li></ol></li></ol>

<ol>
	<li>Wu, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+focal+adhesions+and+barrier+function.">Endothelial focal adhesions and barrier function.</a> J Physiol. 569, 359-366 (2005).</li><li>Geiger, B., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+architecture+and+function+of+matrix+adhesions.">Molecular architecture and function of matrix adhesions.</a> Cold Spring Harb Perspect Biol. 3, (2011).</li><li>Rees, M. D., Kennett, E. C., Whitelock, J. M., Davies, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+damage+to+extracellular+matrix+and+its+role+in+human+pathologies.">Oxidative damage to extracellular matrix and its role in human pathologies.</a> Free Radic Biol Med. 44, 1973-2001 (2008).</li><li>Cox, T. R., Erler, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remodeling+and+homeostasis+of+the+extracellular+matrix:+implications+for+fibrotic+diseases+and+cancer.">Remodeling and homeostasis of the extracellular matrix: implications for fibrotic diseases and cancer.</a> Dis Model Mech. 4, 165-178 (2011).</li><li>Baldus, S., 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=Endothelial+transcytosis+of+myeloperoxidase+confers+specificity+to+vascular+ECM+proteins+as+targets+of+tyrosine+nitration.">Endothelial transcytosis of myeloperoxidase confers specificity to vascular ECM proteins as targets of tyrosine nitration.</a> J Clin Invest. 108, 1759-1770 (2001).</li><li>Baldus, S., 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=Spatial+mapping+of+pulmonary+and+vascular+nitrotyrosine+reveals+the+pivotal+role+of+myeloperoxidase+as+a+catalyst+for+tyrosine+nitration+in+inflammatory+diseases.">Spatial mapping of pulmonary and vascular nitrotyrosine reveals the pivotal role of myeloperoxidase as a catalyst for tyrosine nitration in inflammatory diseases.</a> Free Radic Biol Med. 33, 1010-1019 (2002).</li><li>Thomas, S. R., Witting, P. K., Drummond, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redox+control+of+endothelial+function+and+dysfunction:+molecular+mechanisms+and+therapeutic+opportunities.">Redox control of endothelial function and dysfunction: molecular mechanisms and therapeutic opportunities.</a> Antioxid Redox Signal. 10, 1713-1765 (2008).</li><li>Rees, M. 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=Myeloperoxidase-derived+oxidants+selectively+disrupt+the+protein+core+of+the+heparan+sulfate+proteoglycan+perlecan.">Myeloperoxidase-derived oxidants selectively disrupt the protein core of the heparan sulfate proteoglycan perlecan.</a> Matrix Biol. 29, 63-73 (2010).</li><li>Rees, M. 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=Targeted+subendothelial+matrix+oxidation+by+myeloperoxidase+triggers+myosin+II-dependent+de-adhesion+and+alters+signaling+in+endothelial+cells.">Targeted subendothelial matrix oxidation by myeloperoxidase triggers myosin II-dependent de-adhesion and alters signaling in endothelial cells.</a> Free Radic Biol Med. 53, 2344-2356 (2012).</li><li>Solly, K., Wang, X., Xu, X., Strulovici, B., Zheng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+real-time+cell+electronic+sensing+(RT-CES)+technology+to+cell-based+assays.">Application of real-time cell electronic sensing (RT-CES) technology to cell-based assays.</a> Assay Drug Dev Technol. 2, 363-372 (2004).</li><li>Giaever, I., Keese, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromotion+of+mammalian+cells+measured+electrically.">Micromotion of mammalian cells measured electrically.</a> Proc Natl Acad Sci U S A. 88, 7896-7900 (1991).</li><li>Vissers, M. C., Thomas, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypochlorous+acid+disrupts+the+adhesive+properties+of+subendothelial+matrix.">Hypochlorous acid disrupts the adhesive properties of subendothelial matrix.</a> Free Radic Biol Med. 23, 401-411 (1997).</li><li>Ziv, N. E., Schiller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+Interference+Contrast+(DIC)+Imaging+of+Living+Cells.">Differential Interference Contrast (DIC) Imaging of Living Cells.</a> CSH Protoc. 2007, (2007).</li><li>Kennett, E. C., 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=Peroxynitrite+modifies+the+structure+and+function+of+the+extracellular+matrix+proteoglycan+perlecan+by+reaction+with+both+the+protein+core+and+the+heparan+sulfate+chains.">Peroxynitrite modifies the structure and function of the extracellular matrix proteoglycan perlecan by reaction with both the protein core and the heparan sulfate chains.</a> Free Radic Biol Med. 49, 282-293 (2010).</li><li>Sen, S., Ng, W. P., Kumar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contractility+dominates+adhesive+ligand+density+in+regulating+cellular+de-adhesion+and+retraction+kinetics.">Contractility dominates adhesive ligand density in regulating cellular de-adhesion and retraction kinetics.</a> Ann Biomed Eng. 39, 1163-1173 (2011).</li><li>Wildt, B., Wirtz, D., Searson, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmed+subcellular+release+for+studying+the+dynamics+of+cell+detachment.">Programmed subcellular release for studying the dynamics of cell detachment.</a> Nat Methods. 6, 211-213 (2009).</li></ol>

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使用细胞 - 基底阻抗和活细胞成像分析细胞 - 基质粘附和脱粘附过程可以精确地定量实时地与高时间分辨率。这些实时的方式提供的一大优势终点细胞粘附，提供差的时间分辨率的分析。通过精确量化的快速去应对的粘附具有高时间分辨率，这些分析可以提供重要的见解如何形态响应矩阵修改规定，以及它们如何影响粘附依赖性细胞信号传导过程，在并行使用相关的生化分析仪（ 例如 ， W...

需要进行维护组织稳态的细胞和它们的周围的细胞外基质之间稳定的粘合接触。例如，内皮细胞粘附到内皮下基质中的血管在维持内皮细胞层的完整性和其体内平衡功能的调节，半渗透血管屏障1的关键作用。肌动蛋白细胞骨架被机械地联接到粘合剂基质分子在细胞 - 基质粘附和粘合剂接触，在细胞 - 基质界面部位在确定通过抵抗中心地定向肌动球蛋白的拉力细胞膜的位置中发挥重要作用。该改变的细胞 - 基质粘附性不一定改变力的细胞 - 基质界面的平衡，一个事件，是迅速外刺激&#39;感测&#39;通过机械性敏感信号蛋白，从而产生"外信令"的转导。这种串扰的赌注吐温细胞及其周围的细胞外基质在控制细胞形状，运动性，功能，增殖和存活2关键作用。 多样病理生理过程（胚胎发育，炎症，伤口修复和肿瘤转移）的特征在于粘合剂矩阵基板动态重构由基质降解氧化剂和/或酶3,4。例如，粘合剂在血管内皮下基质蛋白（ 如纤连蛋白）有牵连作为用于修改或降解在人类炎性疾病主要目标，由于反应性的氧化剂的局部生产（ 例如 ，次氯酸，次氯酸）由白细胞衍生的酶髓过氧化物酶（MPO），炎性血管疾病（ 图1）5-9在内皮下膜在其内积聚。诱导的MPO来源的氧化剂和其它基质修饰的刺激的变化的细胞 - 基质粘附是LIK伊利发挥重要的作用在期间各种病理过程的改变血管稳态; 例如 ，通过改变内皮细胞信号传导，形态和生存力，这反过来扰乱内皮功能和屏障的完整性。然而，贴壁细胞向细胞外基质的修改的形态学和细胞信号传导的响应才刚刚开始被理解。 理解矩阵的修改如何驱动改变细胞粘附动力学和粘附依赖性细胞信号传导途径，技术要求准确地定量实时变化在细胞 - 基质粘附性，具有高时间分辨率。在这里，我们描述了互补细胞 - 基底阻抗，并且满足这些标准，并提供​​一个平台，来量化细胞粘附和脱粘附过程在一种非侵入性的方式活细胞成像技术。 我们将展示如何将这些细胞基质阻抗和活细胞成像Appro公司疼痛可容易地用于（ⅰ）监测细胞附着的动力学和扩散（ 即 ， 从头细胞粘附）到天然和改性的矩阵基板和（ii）以测量细胞-基质脱离的动力学（ 即 ，脱密合性）在暴露于基质修改刺激壁细胞。在用xCELLigence细胞 - 基底阻抗生物传感器系统通过在96孔金微电极阵列的表面量化电阻抗提供了细胞 - 基质接触面积的连续测量，并表示这些电阻抗测量值作为"单元索引&#39;，一个无量纲值，该值在很大程度上是正比于细胞-基板接触10（ 图2）的面积，同时也被敏感的变化（绝缘）细胞膜和电极表面11之间的平均距离。在细胞指数值的进一步增加也对形成紧密的细胞与细胞的接触塔实现吨旁限制电流流过，11条件并不在这项研究中所描述的实验中占上风。单个细胞随时间的通过时间推移微分干涉对比图像分析投影面积的测量（DIC）的电影提供改变细胞 - 基板接触的面积的一种辅助措施，并提供了有关的确切性质和动态的附加信息由细胞 - 基底阻抗的方法定量的形态学变化。 具体地讲，我们描述了这些方法的应用程序来监视如何MPO介导的内皮下粘合剂基质蛋白（ 如纤连蛋白）（ⅰ）氧化减少悬浮内皮细胞的从头粘附到精制纤连蛋白和（ii）引发的细胞-基质德-adhesion内皮细胞与纤连蛋白的粘附建立。通过执行并行细胞信号分析一段时间内使用相关的生化的iCal测定（ 例如 ，Western印迹），在密合性依赖性细胞信号事件的粘附/解密合性的过程和相关的变化之间的时间和因果关系可以被确定。 这些方法最近被用于证明外基质氧化MPO的内皮下沉积催化触发内皮细胞是受预先存在肌动球蛋白收缩力9驱动的细胞-基质粘附性迅速丧失。重要的是，通过使在两个细胞粘附的变化和粘附依赖性细胞信号被确定之间的时间关系，这些方法识别出的MPO诱导的基质修饰和细胞脱密合触发器变化重要的粘附依赖性细胞信号传导途径，包括Src激酶依赖性桩蛋白的磷酸化和肌球蛋白轻链磷酸二（ 图1）9。的氧化还原 - 依赖性信号，INV此模式通过扰乱细胞-基质粘附细胞外氧化反应olving细胞内信号传导事件的活化，代表了一种新的小区的模式中的信令称为"从外到内氧化还原信号"（ 图1）9。 在一般情况下，这些辅助细胞基质阻抗传感器和活细胞成像的方法应该是有价值的揭示基修饰的刺激或代理商如何不同的驱动改变细胞粘附动态，形态和不同的贴壁细胞类型受到各种各样的内信号实验的设置。 以下方案描述了如何量化上从头内皮细胞粘附（ 实验1）和内皮细胞的去粘合性（ 实验2）过程的MPO介导的基质的氧化的影响。 MPO如饥似渴地结合纤连蛋白和其他胶粘剂内皮细胞外基质蛋白和我们ES过氧化氢 ​​（H 2 O 2），氯离子（Cl -的）转换为高反应性的氯化的氧化剂次氯酸（次氯酸），其局部反应与这些基质蛋白，破坏其细胞粘合性能（ 图1）8,9， 12。

<table><tbody><tr><td>96 well gold cell-substrate impedance microelectrode array</td><td>ACEA Biosciences / Roche</td><td>E-Plate 96</td><td>single-use plate used for performing cell-based assays on the xCELLigence system</td></tr><tr><td>blebbistatin</td><td>Sigma</td><td>B0560</td><td>selective inhibitor of non-muscle myosin-II</td></tr><tr><td>Bovine Aortic Endothelial Cells, cryopreserved&amp;nbsp;</td><td>Lonza</td><td>BW-6001</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>05470</td><td></td></tr><tr><td>EGM-2 BulletKit</td><td>Lonza</td><td>CC-3162</td><td>endothelial cell growth media kit</td></tr><tr><td>Fibronectin, lyophilized powder</td><td>Sigma</td><td>F-4759</td><td>from bovine plasma</td></tr><tr><td>Fluorodish 35 mm glass-bottomed cell culture dish</td><td>World Precision Instruments</td><td>FD35-100</td><td>Cell cuture dish with optical quality glass bottom for imaging</td></tr><tr><td>Gelatin from bovine skin</td><td>Sigma</td><td>G9391</td><td>cell culture substratum</td></tr><tr><td>Hank&#039;s Balanced Salt Solution</td><td>Life Technologies</td><td>14025076</td><td></td></tr><tr><td>Hydrogen peroxide</td><td>Merck</td><td>107298</td><td></td></tr><tr><td>Medium-199</td><td>Life Technologies</td><td>11150-059</td><td>serum-free cell media</td></tr><tr><td>Methionine</td><td>Sigma</td><td>M9500</td><td>quenches chlorinating oxidants generated by myeloperoxidase</td></tr><tr><td>Myeloperoxidase, Human Polymorphonuclear Leukocytes</td><td>Millipore</td><td>475911</td><td></td></tr><tr><td>RTCA MP Instrument / xCELLigence cell substrate impedance system</td><td>ACEA Biosciences / Roche</td><td></td><td>Consists of RTCA Analyzer, RTCA (Multiple Plate) MP Station and RTCA Control Unit</td></tr><tr><td>Trypsin-EDTA (0.5%)</td><td>Gibco</td><td>15400-054&amp;nbsp;</td><td></td></tr></tbody></table>

using cell-substrate impedance and live cell imaging to measure real-time changes in cellular adhesion and de-adhesion induced by matrix modification

细胞 - 基质粘附在控制细胞形态和信令关键作用。扰乱细胞-基质的粘附（ 例如 ，髓过氧化物酶和其它基质改性氧化剂/炎症过程中释放的酶）的刺激有牵连的触发在许多疾病中的细胞功能，表型和生存能力病理变化。在这里，我们描述了如何细胞 - 基底阻抗和活细胞成像的方法可以容易地用于精确定量细胞粘附和脱密合性诱导基质改性实时变化（使用内皮细胞和髓过氧化物酶作为病理生理基质改性刺激）具有高时间分辨率和以非侵入性方式。在用xCELLigence细胞 - 基底阻抗系统连续地通过测量电阻抗在细胞 - 基底界面上生长的金微电极阵列的细胞量化细胞 - 基质粘附的面积。图像分析时间推移differenTiAl金属干涉对比电影量化改变各个单元的投影面积随着时间的推移，代表改变的细胞 - 基质接触的区域。这两种技术准确量化的迅速变化，以细胞粘附和脱粘附过程。在微阵列生物传感器细胞基质阻抗提供了一个平台，为强大的高通量测量。活细胞成像分析提供关于通过细胞基质阻抗测量量化形态变化的性质和动态的更多细节。这些互补的方法提供了宝贵的新的见解如何髓过氧化物酶催化氧化亚细胞外基质成分的改变触发细胞黏附，形态的快速变化和血管内皮细胞的信号。这些方法也适用于研究细胞粘附动力学响应于其它基质修饰的刺激以及相关贴壁细胞（ 例如，上皮细胞）。

内皮细胞从纤连蛋白响应于MPO介导的纤连蛋白氧化（实验2）的内皮细胞悬浮液。将播种到天然酶（MPO免费）纤连蛋白或MPO承载纤连蛋白的结果在最大细胞附着和铺展脱密合性的实时定量在2个小时，作为判断细胞指数值的中电池基板的阻抗测量（ 图3A）一个趋于稳定。该初始细胞附着和扩散的相位显着降低时，MPO介导的纤连蛋白的氧化是在根据?...

Watch this Scientific Journal Video about Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification at JoVE.

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

这里，我们提出了一个协议，通过细胞 - 基底阻抗和活细胞成像分析，以连续地量化具有高时间分辨率的细胞粘附和脱粘附过程以非侵入性方式。这些方法揭示了通过矩阵修饰和它们的粘附依赖性信号事件的时间关系触发细胞粘附/去粘附过程的动力学。

使用Cell-基板阻抗和活细胞成像，以衡量矩阵修改诱导实时变化的细胞粘附和去粘附

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

using-cell-substrate-impedance-live-cell-imaging-to-measure-real-time

Research

JoVE Journal

Bioengineering

10.9K 次观看.  University of New South Wales. 这里，我们提出了一个协议，通过细胞 - 基底阻抗和活细胞成像分析，以连续地量化具有高时间分辨率的细胞粘附和脱粘附过程以非侵入性方式。这些方法揭示了通过矩阵修饰和它们的粘附依赖性信号事件的时间关系触发细胞粘附/去粘附过程的动力学。 

视频: Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

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

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

活细胞成像的移植细胞在一个三维矩阵表示荧光标记的蛋白质

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. To this end, cells are grown in special culture chambers on top of opposing, circular gold electrodes. A constant small alternating current is applied between the electrodes and the potential across is measured. The insulating properties of the cell membrane create a resistance towards the electrical current flow resulting in an increased electrical potential between the electrodes. Measuring cellular impedance in this manner allows the automated study of cell attachment, growth, morphology, function, and motility. Although the ECIS measurement itself is straightforward and easy to learn, the underlying theory is complex and selection of the right settings and correct analysis and interpretation of the data is not self-evident. Yet, a clear protocol describing the individual steps from the experimental design to preparation, realization, and analysis of the experiment is not available. In this article the basic measurement principle as well as possible applications, experimental considerations, advantages and limitations of the ECIS system are discussed. A guide is provided for the study of cell attachment, spreading and proliferation; quantification of cell behavior in a confluent layer, with regard to barrier function, cell motility, quality of cell-cell and cell-substrate adhesions; and quantification of wound healing and cellular responses to vasoactive stimuli. Representative results are discussed based on human microvascular (MVEC) and human umbilical vein endothelial cells (HUVEC), but are applicable to all adherent growing cells.

This protocol reviews Electric Cell-substrate Impedance Sensing, a method to record and analyze the impedance spectrum of adherent cells for the quantification of cell attachment, proliferation, motility, and cellular responses to pharmacological and toxic stimuli. Detection of endothelial permeability and assessment of cell-cell and cell-substrate contacts are emphasized.

电动细胞基质阻抗传感的内皮细胞增殖，屏障功能的定量和能动性

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. ...

Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These acquired abnormalities in complex, multilayered molecular signaling networks induce aberrant cell proliferation and survival, extracellular matrix degradation, and metastasis to distant organs. Approximately 90% of cancer-related deaths are estimated to be caused by the direct or indirect effects of metastatic dissemination. Therefore, it is important to establish a highly reliable, comprehensive system to characterize cancer cell behaviors upon genetic and environmental manipulations. Such a system can give a clear understanding of the molecular regulation of cancer metastasis and the opportunity for successful development of stratified, precise therapeutic strategies. Hence, accurate determination of cancer cell behaviors such as migration and invasion with gain or loss of function of gene(s) allows assessment of the aggressive nature of cancer cells. The real-time measurement system based on cell impedance enables researchers to continually acquire data during a whole experiment and instantly compare and quantify the results under various experimental conditions. Unlike conventional methods, this method does not require fixation, staining, and sample processing to analyze cells that migrate or invade. This method paper emphasizes detailed procedures for real-time determination of migration and invasion of glioblastoma cancer cells.

Cancer is a lethal disease due to its ability to metastasize to different organs. Determining the ability of cancer cells to migrate and invade under various treatment conditions is crucial to assessing therapeutic strategies. This protocol presents a method to assess the real-time metastatic abilities of a glioblastoma cancer cell line.&#160;

基于阻抗的癌细胞迁移和入侵的实时测量

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These ...

癌症研究

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

在一个三维胶原蛋白矩阵分析细胞迁移

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to tumor and cancer metastasis. Despite intense research efforts, the basic biochemical and biophysical principles of cell migration are still not fully understood, especially in the physiologically relevant three-dimensional (3D) microenvironments. Here, we describe an in vitro assay designed to allow quantitative examination of 3D cell migration behaviors. The method exploits the cell&#8217;s mechanosensing ability and propensity to migrate into previously unoccupied extracellular matrix (ECM). We use the invasion of highly invasive breast cancer cells, MDA-MB-231, in collagen gels as a model system. The spread of cell population and the migration dynamics of individual cells over weeks of culture can be monitored using live-cell imaging and analyzed to extract spatiotemporally-resolved data. Furthermore, the method is easily adaptable for diverse extracellular matrices, thus offering a simple yet powerful way to investigate the role of biophysical factors in the microenvironment on cell migration.

The mechanical properties and microstructure of the extracellular matrix strongly affect 3D migration of cells. An in vitro method to study the spatiotemporal cell migration behavior in biophysically variable environments, at both population and individual cell levels, is described.

同心凝胶系统来研究基质微环境的三维细胞迁移的生物物理作用

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to ...

Real-Time Detection and Capture of Invasive Cell Subpopulations from Co-Cultures

Invasion and metastatic spread of cancer cells are the major cause of death from cancer. Assays developed early on to measure the invasive potential of cancer cell populations typically generate a single endpoint measurement that does not distinguish between cancer cell subpopulations with different invasive potential. Also, the tumor microenvironment consists of different resident stromal and immune cells that alter and participate in the invasive behavior of cancer cells. Invasion into tissues also plays a role in immune cell subpopulations fending off microorganisms or eliminating diseased cells from the parenchyma and endothelial cells during tissue remodeling and angiogenesis. Real-Time Cellular Analysis (RTCA) that utilizes impedance biosensors to monitor cell invasion was a major step forward beyond endpoint measurement of invasion: this provides continuous measurements over time and thus can reveal differences in invasion rates that are lost in the endpoint assay. Using current RTCA technology, we expanded dual-chamber arrays by adding a further chamber that can contain stromal and/or immune cells and allows measuring the rate of invasion under the influence of secreted factors from co-cultured stromal or immune cells over time. Beyond this, the unique design allows for detaching chambers at any time and isolating of the most invasive cancer cell, or other cell subpopulations that are present in heterogeneous mixes of tumor isolates tested. These most invasive cancer cells and other cell subpopulations drive malignant progression to metastatic disease, and their molecular characteristics are important for in-depth mechanistic studies, the development of diagnostic probes for their detection, and the assessment of vulnerabilities. Thus, the inclusion of small- or large-molecule drugs can be used to test the potential of therapies that target cancer and/or stromal cell subpopulations with the goal of inhibiting (e.g., cancer cells) or enhancing (e.g., immune cells) invasive behavior.

We describe an approach to detect and capture invasive cell subpopulations in real-time. The experimental design uses Real-Time Cellular Analysis by monitoring changes in the electric impedance of cells. Invasive cancer, immune, endothelial or stromal cells in complex tissues can be captured, and the impact of co-cultures can be assessed.

实时检测和捕获共培养物中的侵袭细胞亚群

Invasion and metastatic spread of cancer cells are the major cause of death from cancer. Assays developed early on to measure the invasive potential of ...

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

机械性刺激，集成光学和原子力显微镜研究活细胞的反应

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

生物学

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

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.