The Extracellular Matrix (ECM) is composed of collagen, non-collagenous glycoproteins and proteoglycans. These components are secreted from cells to create an ECM meshwork that surrounds cells' and tissues. The ECM regulates many aspects of cellular function, including the cells dynamic behavior, cytoskeletal organization and intercellular communication.
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Question 1: Can I coat cells with fluorescent ECM proteins?
Answer 1: Yes, cells can be coated with fluorescently-labeled ECM proteins such as fibronectin. Example protocols are found in Pallis et al., 1997 (Peripheral Blood Lymphocyte Binding to a Soluble FITC-Fibronectin Conjugate. Cytometry. 28, 157-164) and Huveneers et al., 2008 (Binding of soluble fibronectin to integrin a5b1 – link to focal adhesion redistribution and contractile shape. J. Cell Sci. 121, 2452-2462). Briefly, cells are incubated with a low concentration of soluble fluorescently-labeled fibronectin in an appropriate buffer. Low concentrations of fibronectin are recommended to minimize non-specific binding. The cells/fibronectin mixture is incubated in the dark for 30 min at room temperature. Others have done the incubation at 4°C for 1 hour. The cells are then rinsed in an appropriate buffer, resuspended in the same buffer, and then the coating/binding of ECM proteins can be quantified by flow cytometry or the coated cells can be used experimentally.