title: A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix.
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.
authors.affiliation: School of Biochemistry, University of Bristol.
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Hellewell
authors.affiliation: School of Biochemistry, University of Bristol.
authors.forename: Silvia
authors.initials: S
authors.lastname: Rosini
authors.affiliation: School of Biochemistry, University of Bristol; Jo.Adams@bristol.ac.uk.
authors.forename: Josephine C
authors.initials: JC
authors.lastname: Adams
mesh_terms: D064753:Ammonium Hydroxide / Q000737:chemistry; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D004591:Electrophoresis, Polyacrylamide Gel; D005109:Extracellular Matrix / Q000737:chemistry* / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D059008:Time-Lapse Imaging; D014508:Urea / Q000737:chemistry
chemical_list: D016326:Extracellular Matrix Proteins; D064753:Ammonium Hydroxide; D014508:Urea