Abstract
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.
Highlights
Over the last few decades, the extracellular matrix (ECM) has become recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes
The protocol was carried out on COS-7 cells grown for 96 h on glass coverslips, and the cell-derived ECM was analyzed by fluorescence microscopy
Ammonium hydroxide is stable in aqueous solutions in the dark and can be stored at room temperature, bottles must be tightly re-sealed between each use
Summary
Over the last few decades, the extracellular matrix (ECM) has become recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. Cell-derived ECM is composed of many large, oligomeric proteins, which are often covalently cross-linked upon ECM assembly and are insoluble in standard detergents. These properties can complicate extraction and the further analysis of ECM. Several methods have been described in the literature for the isolation of ECM from either cell culture or tissue extracts. Many of these methods are aimed at the extraction of the abundant ECM protein, collagen, from tissues and include the use of neutral salts[3], acidic conditions[5,6], or pepsin[7]. Www.jove.com approach provides an accurate isolation of cell-derived ECM and the scope to identify and monitor the deposition and dynamics of individual ECM proteins
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