Departamento de Bioquímica, Universidad Autónoma de Madrid, Instituto de Investigaciones Biomédicas 'Alberto Sols' CSIC-UAM, Madrid, Spain.
Nat Protoc. 2009;4(11):1591-613. doi: 10.1038/nprot.2009.152. Epub 2009 Oct 15.
Here we describe several methods for the characterization of epithelial-mesenchymal transition (EMT) at the cellular, molecular and behavioral level. This protocol describes both in vitro and in vivo approaches designed to analyze different features that when taken together permit the characterization of cells undergoing transient or stable EMT. We define straightforward methods for phenotypical, cellular and transcriptional characterization of EMT in vitro in monolayer cultures. The procedure also presents technical details for the generation of in vitro three-dimensional (3D) cultures analyzing cell phenotype and behavior during the EMT process. In addition, we describe xenotransplantation techniques to graft 3D cell cultures into mice to study in vivo invasion in a physiological-like environment. Finally, the protocol describes the analysis of selected EMT markers from experimental and human tumor samples. This series of methods can be applied to the study of EMT under various experimental and biological situations. Once the methodology is established, the time required to complete the protocol may vary from 3 to 4 weeks (monolayer cultures) and up to 6-8 weeks if including 3D cultures.
在这里，我们描述了几种用于在细胞、分子和行为水平上表征上皮-间充质转化 (EMT) 的方法。该方案描述了旨在分析不同特征的体外和体内方法，这些特征综合起来可用于表征经历短暂或稳定 EMT 的细胞。我们定义了用于体外单层培养中 EMT 的表型、细胞和转录特征分析的直接方法。该程序还介绍了用于生成体外三维 (3D) 培养物的技术细节，以在 EMT 过程中分析细胞表型和行为。此外，我们描述了将 3D 细胞培养物移植到小鼠中以在生理样环境中研究体内侵袭的异种移植技术。最后，该方案描述了从实验和人类肿瘤样本中分析选定的 EMT 标志物。这一系列方法可应用于各种实验和生物学情况下的 EMT 研究。一旦建立了方法学，完成该方案所需的时间可能从 3 到 4 周（单层培养物）不等，如果包括 3D 培养物，则最多需要 6-8 周。
