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
Summary
我们描述了典型的方法涂有荧光可视化侵袭伪足介导的基质降解明胶的生产显微镜的盖玻片。利用现有的软件的计算技术，提出了一个混合的人口和量化结果由单个细胞内的基质蛋白水解为多细胞群体，涵盖整个微观领域。
Abstract
局部组织的细胞浸润到是一个重要的发展和平衡的过程。 malregulated入侵并随后细胞运动是多个病理过程，包括炎症，心血管疾病和肿瘤细胞转移1的特征。 focalized蛋白水解降解的胞外基质（ECM）的组件中的上皮细胞或内皮基底膜是启动细胞侵袭的一个关键步骤。在肿瘤细胞中，已确定， 在体外分析广泛ECM的降解是通过腹侧肌动蛋白富集膜凸结构称为侵袭伪足2,3。侵袭伪足形式在紧密合到的ECM，在那里它们通过的作用，基质金属蛋白酶（MMPs）中度ECM击穿。肿瘤细胞的能力，以形成侵袭伪足直接相关与标注侵入本地基质和相关的血管成分3。
_content“>可视化的侵袭伪足介导的细胞外基质降解的细胞荧光显微镜用染料标记的基质蛋白涂在玻片上已经成为最流行 的技术评估基质蛋白水解的程度和细胞侵袭能力4,5。在这里，我们描述一个版本产生荧光标记的玻片上，利用商业可用的俄勒冈绿-488明胶共轭的标准方法，这种方法很容易扩展到迅速产生大量的涂盖玻片，我们中经常遇到的一些常见的微观文物在此过程中，如何将这些可避免的。最后，我们使用现成的电脑软件，允许标记的明胶基质的降解介导的单个细胞和整个细胞群的量化描述的标准化方法。所描述的程序提供了准确和可重复的监测能力侵袭伪足活性，也可以作为一个平台，用于评估调制蛋白的表达或测试抗侵入性的胞外基质降解的化合物对单和多细胞设置的疗效。Protocol
1。俄勒冈州绿色生产488-明胶涂层盖玻片准备一个未标记的5％（w / w的）库存明胶/蔗糖溶液加入1.25克明胶和1.25克蔗糖在PBS中至终体积为50ml。的股票明胶溶液预热至37°C，并确保它完全融化之前使用。最终混合物在4℃下存放清洁直径为13mm的第1玻璃盖玻片放置到24孔塑料组织培养板的各孔中的一个单独的盖玻片。向每孔中加入500μl的20％硝酸中，并温育30分钟。吸液的硝酸溶液，并用…
Discussion
辅助能力，可视化细胞降解细胞外基质中发现的早期步骤侵袭细胞的分子机制。陈文添在20世纪80年代初4,14,15首创，涂料荧光标记的细胞外蛋白质随后的微观分析的盖玻片上已经出现的主要技术评估侵袭伪足的功能范围广泛的细胞类型。规定的协议演示的基本的方法，用于制备明胶包被的盖玻片，形成一个小于2微米厚的胶原层适合于检测到的细胞外基质降解由细胞中最常规的荧光和激光?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是从西弗吉尼亚大学玛丽巴布伦道夫癌症中心捐赠基金的支持。我们感谢“穆勒Susette（乔治城大学）和劳拉·凯利早期的意见和协助。西弗吉尼亚大学的显微成像设备的使用（支持玛丽巴布兰多夫癌症中心，国立卫生研究院资助P20 RR16440，P30 RR032138和P30 GM103488）表示感谢。
Materials
| Name of Reagent/Instrument | Company | Catalogue Number | Comments |
| gelatin | Sigma | G1890 | Porcine skin |
| sucrose | Fisher | BP220 | |
| 12 mm coverslips | Fisher | 50-121-5159 | |
| 24 well plates | Fisher | 08-772-1 | BD Falcon |
| nitric acid | Ricca | R5326000 | 20% solution |
| poly-L-lysine | Electron Microscopy Sciences | 19320-B | 0.1% solution |
| glutaraldehyde | Sigma | G7526 | 8% solution |
| Oregon Green 488-conjugated gelatin | Invitrogen | G-13186 | |
| sodium borohydride | Sigma | 213462 | |
| Triton X-100 | Fisher | BP151 | |
| rhodamine-phalloidin | Invitrogen | R415 | |
| anti-cortactin (clone 4F11) | Millipore | 05-180 | |
| Anti-FLAG antibody | Millipore | MAB3118 | |
| Alexa Fluor 647 Goat anti-mouse IgG | Invitrogen | A21235 | |
| ProLong Gold antifade | Invitrogen | P36930 | |
| LSM 510 Confocal Microscope | Zeiss | ||
| ImageJ software | Public domain | http://www.macbiophotonics.ca/imagej/ | |
| AutoQuant X2.2 software | Media Cybernetics | ||
| NIS Elements software | Nikon |
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Tags
Quantitative MeasurementInvadopodia-mediatedExtracellular Matrix ProteolysisSingle And Multicellular ContextsCellular InvasionDevelopmentHomeostasisMalregulated InvasionCell MovementPathological ProcessesInflammationCardiovascular DiseaseTumor Cell MetastasisProteolytic DegradationExtracellular Matrix (ECM)Epithelial Basement MembraneEndothelial Basement MembraneVentral Actin-rich Membrane Protrusive StructuresInvadopodiaMatrix Metalloproteinases (MMPs)Tumor CellsIn Vitro AnalysisECM DegradationFluorescent MicroscopyDye-labeled Matrix ProteinsGlass CoverslipsEvaluating Matrix ProteolysisCellular Invasive Potential
Cite This Article
Martin, K. H., Hayes, K. E., Walk, E. L., Ammer, A. G., Markwell, S. M., Weed, S. A. Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts. J. Vis. Exp. (66), e4119, doi:10.3791/4119 (2012).