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
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?...

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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 border="0" cellpadding="0" cellspacing="0" width="1328">
	<tbody>
		<tr height="21">
			<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?...

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 ...

Research

JoVE Journal

Bioengineering

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

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

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

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

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

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 ...

Hand-held Clinical Photoacoustic Imaging System 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

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

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) ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

用于研究基质细胞代谢串扰的人类3D细胞外基质-脂肪培养模型

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...