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>

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作者没有披露作出。

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 ， Western印迹）。 在最近的研究中，这些方法被用于揭示在触发内皮细胞的脱密合性的内皮下矩阵的MPO介导的氧化作用的新型作用并表现出:（i）该解密合性的比率是严格依赖于预现有的肌动球蛋白收缩（参见图3B和3C）和（ii），该细胞-基质粘附性的损失开车变化重要的粘附依赖性细胞信号传导途径，包括Src蛋白依赖性桩蛋白的磷酸化和肌球蛋白轻链磷酸二9（ 见图1）。这些数据对过程中的炎症反应，其中内皮细胞外基质被牵连由基质结合MPO或活性氧化剂3,5-9其他来源的内皮下沉积产生氧化剂的主要目标理解内皮功能和屏障的完整性具有重要意义， 14。 使用微阵列为基础的，细胞 - 基底阻抗生物传感器系统的提供健壮，高通量细胞粘附测量的平台。在96孔细胞-基底阻抗微电极阵列的每个孔中有同时分析32个不同的实验条件（ 即 ，3个孔PE的电位ř条件）。但是，比较在细胞粘附的微小变化时，至少4个或更多个井应每个实验条件下使用。在细胞接种和随后的细胞的治疗，必须小心，以保持所有的解决方案，在37℃，并尽量减少治疗37℃培养箱环境以外的细胞（这避免了在整个微电极阵列，可能会影响粘合性的响应潜在的温度差所用的时间）。值得注意的是，在细胞-基底界面的电流是向细胞上清液10的离子强度敏感:因此，细胞应允许增加新的缓冲器/媒体的后平衡监测效果之前，以获得一个稳定的细胞指数反应感兴趣的刺激（见3.10和图3A）。减小小区索引不仅可以反映在细胞 - 基质接触的平均面积的减少，但也可能反映在附着的细胞的数目减少。歧视这些可能性之间的变化，在微电极阵列附着的细胞的数目可以立即后通过结晶紫染色这一方法已被用于确认的MPO介导的纤连蛋白氧化触发中的平均面积迅速下降细胞 - 基底阻抗测量量化细胞-基质接触，但并没有促进细胞支队（有关详细信息，请参阅第9）。 在细胞 - 基底阻抗技术的一个限制是，它提供了超过存在于微电极表面上的所有细胞的粘附的变化的平均量度，它不给出关于密合性，或形态学改变的确切性质在单个细胞水平的信息。为了解决这个限制，活细胞DIC显微镜和图像分析提供了附着力变化互补措施，并揭示了单个细胞的形态变化。因此，降低细胞指数为响应MPO介导的纤维连接蛋白氧化MEA由细胞-基底阻抗（ 图3A），在由活细胞图像的DIC电影分析（ 图3B）测量细胞的投影面积的下降相关性良好sured。重要的是，DIC电影揭示的是，在单个细胞，MPO诱导的脱附着力涉及到从底层及相邻小区的周边细胞膜的快速缩回，并最终导致在细胞中假设一个紧凑的"舍入式"的形态，但没有得到在细胞脱离（ 电影1）。与细胞基质阻抗测量，以确保可重复的细胞反应，应注意以维护解决方案37°C到对细胞和温水显微镜阶段（或同等温度控制装置）必须在活细胞成像过程中采用使用前录音。 这里描述的方案可以容易地通过与其他纯化的矩阵基板代纤连蛋白（被修改<em&gt;如胶原蛋白或层粘连蛋白）或培养的贴壁细胞研究一种更复杂的矩阵环境中产生的加班（详见9）。该协议也可以通过与扰乱细胞-基质粘附其它可溶性刺激包括其他生理基质改性氧化剂（ 例如 ，过氧化亚硝酸盐，二氧化氮自由基5-8），基质降解蛋白酶15或拮抗剂代MPO介导的基质氧化改变存在在细胞表面上或在基质（ 例如 ，抗整合素抗体或RGD肽）粘合剂的配体。活细胞成像的分析也可以被用于量化细胞-基质脱的附着力响应于细胞-基质触点16的电化学解吸。最后，所描述的协议也容易适用于研究细胞粘附动力学在贴壁细胞比内皮细胞（ 例如 ，上皮细胞）等。

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。

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			<td height="21" style="height:21px;width:405px;">Name of Material/ Equipment</td>
			<td style="width:223px;">Company</td>
			<td style="width:151px;">Catalog Number</td>
			<td style="width:551px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">96 well gold cell-substrate impedance microelectrode array</td>
			<td style="width:223px;">ACEA Biosciences / Roche</td>
			<td style="width:151px;">E-Plate 96</td>
			<td>single-use plate used for performing cell-based assays on the xCELLigence system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">blebbistatin</td>
			<td style="width:223px;">Sigma</td>
			<td style="width:151px;">B0560</td>
			<td>selective inhibitor of non-muscle myosin-II</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Bovine Aortic Endothelial Cells, cryopreserved&#160;</td>
			<td style="width:223px;">Lonza</td>
			<td style="width:151px;">BW-6001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Bovine serum albumin</td>
			<td style="width:223px;">Sigma</td>
			<td style="width:151px;">05470</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGM-2 BulletKit</td>
			<td style="width:223px;">Lonza</td>
			<td style="width:151px;">CC-3162</td>
			<td>endothelial cell growth media kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Fibronectin, lyophilized powder</td>
			<td style="width:223px;">Sigma</td>
			<td style="width:151px;">F-4759</td>
			<td>from bovine plasma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Fluorodish 35 mm glass-bottomed cell culture dish</td>
			<td style="width:223px;">World Precision Instruments</td>
			<td style="width:151px;">FD35-100</td>
			<td>Cell cuture dish with optical quality glass bottom for imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Gelatin from bovine skin</td>
			<td style="width:223px;">Sigma</td>
			<td style="width:151px;">G9391</td>
			<td>cell culture substratum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Hank&#39;s Balanced Salt Solution</td>
			<td style="width:223px;">Life Technologies</td>
			<td style="width:151px;">14025076</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Hydrogen peroxide</td>
			<td style="width:223px;">Merck</td>
			<td style="width:151px;">107298</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Medium-199</td>
			<td style="width:223px;">Life Technologies</td>
			<td style="width:151px;">11150-059</td>
			<td>serum-free cell media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Methionine</td>
			<td style="width:223px;">Sigma</td>
			<td style="width:151px;">M9500</td>
			<td>quenches chlorinating oxidants generated by myeloperoxidase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Myeloperoxidase, Human Polymorphonuclear Leukocytes</td>
			<td style="width:223px;">Millipore</td>
			<td style="width:151px;">475911</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">RTCA MP Instrument / xCELLigence cell substrate impedance system</td>
			<td style="width:223px;">ACEA Biosciences / Roche</td>
			<td style="width:151px;">-</td>
			<td>Consists of RTCA Analyzer, RTCA (Multiple Plate) MP Station and RTCA Control Unit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Trypsin-EDTA (0.5%)</td>
			<td>Gibco</td>
			<td>15400-054&#160;</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实验1中详述的协议进行的实验之前，细胞接种启动的（数据未示出;详见9）。一旦最大细胞的粘附是建立在天然或MPO-轴承纤连蛋白，纤连蛋白靶向氧化MPO催化的次氯酸介导（通过加入MPO的共同底物H的发起2 O 2），使在河畔迅速下降一个代表性的DIC电影，看电影1;由细胞基质阻抗（ 图3A，B）和活细胞成像（ 图3C检测的细胞基质的接触面区域后，H 2 O 2除了细胞指数或单元面积亏损是最小的在没有MPO 9）。虽然在MPO诱导去黏附细胞指数和细胞面积的变化非常相似，在细胞指数值相对较慢的下降可能反映了在DE-中存在的"无细胞"地区电池膜材料在细胞周围的绝缘效果附着力，这未在投影单元面积的测量定量。快速蜂窝脱密合在结合于MPO承载纤连蛋白响应于H 2 O 2处理的内皮细胞明显不存在在用H 2 O 2的单独处理的细胞（ 即 ，细胞不含有MPO）（ 图3A）和受阻BýMPO酶抑制剂或清除剂次氯酸（未示出的数据;见9说明），识别该过程是依赖于MPO催化生产次氯酸（参见图1）。肌球蛋白II的运动功能与II型肌球蛋白抑制抑制由细胞-基底阻抗（ 图3B）和通过活细胞成像（ 图3C，电影2）测得的内皮细胞的去粘附率，确定在响应该蜂窝脱粘附和收缩至MPO催化的亚细胞基质的氧化是由肌动球蛋白的拉力9驱动。 <img alt="图1" src="/files/ftp_upload/52423/52423fig1highres.jpg" /> 图1. MPO介导的细胞内基质的氧化触发细胞-基质脱粘附和细胞信号传导。MPO通过介导靶向ö触发快速脱密合反应和变化的粘附依赖性信号的粘合剂内皮下基质蛋白，涉及以下事件9 xidation:（A）中的MPO贪婪地结合到内皮下矩阵，并使用H 2 O 2的产生的高反应性的氧化剂次氯酸发生反应局部与基质蛋白（ 如纤连蛋白）和破坏其细胞粘合性能。 （B）的这种损伤破坏的细胞-基质界面粘合剂接触，导致（C）的膜内陷通过无对抗的张力，在肌动蛋白细胞骨架和粘附依赖性细胞信号传导途径（从Rees 等许可的使用的改变驱动的。9 ）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52423/52423fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52423/52423fig2highres.jpg" /> 造型玩具细胞-基底阻抗电子2.工作流和原理分析来量化来自纤连蛋白的内皮细胞的去粘合性响应于MPO介导的纤连蛋白氧化（实验2）。由细胞-基底芯片阻抗的生物传感器系统测量细胞指数值反映细胞膜的限制外地诱导离子电流在电极表面是成正比的细胞-基底接触10的区域的能力。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52423/52423fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52423/52423fig3highres.jpg" /> 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）脱密合性的图3的实时定量。内皮细胞悬浮液（在含有无血清培养基199 1％BSA）中接种到天然或MPO-轴承纤连蛋白（纤连蛋白涂覆的5微克/毫升，然后在96孔的微电极阵列（细胞 - 基底阻抗测量）或35毫米的玻璃底的情况下，或在20纳米的MPO存在）温育细胞培养皿（活细胞成像分析）。然后将细胞温育在37℃下2小时，以允许最大的细胞附着和平衡的细胞用HBSS和发起的MPO介导的纤连蛋白的氧化通过添加H 2 O 2（10μM）的H 2 O 2的加成设定在（时间之前扩频T = 0分钟）。 （A） 的全部时间过程之前和用H 2 O 2中存在的（+ MPO）和不存在的MPO（-MPO）处理后的细胞-底物阻抗测量。 （B）细胞衬底阻抗测量和（C）单元面积测量由活细胞成像分析，处理含MPO细胞与H 2 O 2的存在（后+ II型肌球蛋白）和肌球蛋白II抑制剂II型肌球蛋白（40μM）的情况下（-blebbistatin）。单元索引和小区区域的值被标准化为值之前立即的H 2 O 2的加入时间，其被赋予的1.细胞的索引数据的值表示平均值±SEM，来自一个代表性实验每组6重复测量。小区区域值表示平均值±SEM，每组10细胞（2复制电影，每部电影5随机选择的细胞:看电影1和2）。 （ 图3B，图3C，电影1和 Movie 2由里斯等人 9授权转载）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52423/52423fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52423/Movie 1.mov">电影1</a> 。时间流逝DIC微附着于MPO-轴承纤连超过0内皮细胞镜检查电影-在暴露于H 2 O 2（10μM）15分钟; 1秒= 3分钟。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52423/Movie 2.mov">电影2</a> 。附着于MPO承载纤连超过0内皮细胞的时间推移DIC显微镜电影-在暴露于H 2 O 2（10μM）在II型肌球蛋白（40μM）的存在下15分钟; 1秒= 3分钟。

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

Nanyang Technological University

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

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 ...

科研

生物工程

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

收获小鼠肺泡巨噬细胞和评价聚酸酐纳米粒子诱导的细胞活化

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

Simultaneously Capturing Real-time Images in Two Emission Channels Using a Dual Camera Emission Splitting System: Applications to Cell Adhesion

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to investigate mechanisms governing cell adhesion under dynamic flow conditions. For instance, cancer cells labeled with multiple fluorophores can be perfused over a potentially reactive substrate to model mechanisms of cancer metastasis. However, multi-channel single camera systems and color cameras exhibit shortcomings in image acquisition for real-time live cell analysis. To overcome these limitations, we used a dual camera emission splitting system to simultaneously capture real-time image sequences of fluorescently labeled cells in the flow chamber. Dual camera emission splitting systems filter defined wavelength ranges into two monochrome CCD cameras, thereby simultaneously capturing two spatially identical but fluorophore-specific images. Subsequently, psuedocolored one-channel images are combined into a single real-time merged sequence that can reveal multiple target molecules on cells moving rapidly across a region of interest.

Dual camera emission splitting systems for two-color fluorescence microscopy generate real-time image sequences with exceptional optical and temporal resolution, a requirement of certain live cell assays including parallel plate flow chamber adhesion assays. When software is employed to merge images from simultaneously acquired emission channels, pseudocolored image sequences are produced.

同时捕获实时图像在两个发射通道采用了双摄像头发射分光系统:应用到细胞粘附

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

使用微流体芯片实时成像和损伤研究的响应<em&gt;果蝇</em&gt;幼虫

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

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. ...

Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously attaining anatomical, functional and molecular contrast in deep optically opaque tissues. While enormous potential has been recently demonstrated in the application of optoacoustics for small animal research, vast efforts have also been undertaken in translating this imaging technology into clinical practice. We present here a newly developed optoacoustic tomography approach capable of delivering high resolution and spectrally enriched volumetric images of tissue morphology and function in real time. A detailed description of the experimental protocol for operating with the imaging system in both hand-held and stationary modes is provided and showcased for different potential scenarios involving functional and molecular studies in murine models and humans. The possibility for real time visualization in three dimensions along with the versatile handheld design of the imaging probe make the newly developed approach unique among the pantheon of imaging modalities used in today&#8217;s preclinical research and clinical practice.

We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice.

通用型手持式三维光声成像探测深部组织人力造影和功能的临床前研究中实时

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously ...

Live Cell Imaging during Mechanical Stretch

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in their capability for measuring responses in real time in live cells or viable tissue. A protocol was developed with the use of a cell actuator to distend live cells grown on or tissues attached to an elastic substrate while imaging with confocal and atomic force microscopy (AFM). Preliminary studies show that tonic stretching of human bronchial epithelial cells caused a significant increase in the production of mitochondrial superoxide. Moreover, using this protocol, alveolar epithelial cells were stretched and imaged, which showed direct damage to the epithelial cells by overdistention simulating one form of lung injury in vitro. A protocol to conduct AFM nano-indentation on stretched cells is also provided.

A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy.

在机械拉伸活细胞成像

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in ...

Hand-held Clinical Photoacoustic Imaging System for Real-time Non-invasive Small Animal Imaging

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, we report a combined photoacoustic and clinical ultrasound imaging system by integrating an ultrasound probe with light delivery for small animal imaging. We demonstrate this by showing sentinel lymph node imaging in small animals along with minimally invasive real-time needle guidance. A clinical ultrasound platform with access to raw channel data allows the integration of photoacoustic imaging leading to a handheld real-time clinical photoacoustic imaging system. Methylene blue was used for sentinel lymph node imaging at 675 nm wavelength. Additionally, needle guidance with dual modal ultrasound and photoacoustic imaging was shown using the imaging system. Depth imaging of up to 1.5 cm was demonstrated with a 10 Hz laser at a photoacoustic imaging frame rate of 5 frames per second.

A clinical handheld photoacoustic imaging system will be demonstrated for real-time non-invasive small animal imaging.

手持式实时非侵入性小动物成像的临床光声成像系统

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, ...

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 ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

三维水凝胶中全球细胞基质金属蛋白酶和代谢活性的测定

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...