A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix.
A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix.
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DOI：
10.3791/55051
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发表时间：
2017-01-04
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影响因子：
--
通讯作者：
Adams JC
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文献类型：
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作者：
Hellewell AL;Rosini S;Adams JC
细胞外基质（ECM）被认为是一个多样、动态且复杂的环境，涉及多种细胞生理和病理过程。然而，从组织或细胞培养物中分离ECM因组装后的ECM具有不溶性和交联性质，以及ECM提取物可能被细胞表面和细胞内蛋白质污染而变得复杂。在此，我们描述了一种用于培养细胞的方法，该方法快速且能可靠地去除细胞以分离出细胞衍生的ECM用于下游实验。
通过使用这种方法，分离出的ECM及其成分可通过原位免疫荧光显微镜观察到。在去除细胞前后，可通过荧光显微镜追踪标记蛋白的沉积来跟踪特定ECM蛋白的动态变化。或者，可提取分离出的ECM用于生化分析，例如十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）和免疫印迹。在更大规模上，可通过质谱对分离出的ECM进行全面的蛋白质组学分析。通过在无菌条件下进行ECM分离，可为任何感兴趣的细胞进行功能或表型研究获得无菌的ECM层。该方法可应用于任何贴壁细胞类型，相对容易操作，并可与多种实验设计相关联。
The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation. Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.