title: Quantifying cellular adhesion to covalently immobilized extracellular matrix proteins by single-cell force spectroscopy.
abstract: Atomic force microscopy (AFM)-based single-cell force spectroscopy (SCFS) enables the quantitative study of cell adhesion under physiological conditions. SCFS probes adhesive interactions of single living cells with substrates such as extracellular matrix (ECM) proteins and other cells. Here, we present a protocol to quantitatively study the adhesion of HeLa cells to covalently immobilized fibronectin and Matrigel™ using SCFS. We describe procedures for (a) functionalization of AFM cantilevers, (b) preparation of maleic anhydride copolymer thin films, (c) covalent immobilization of ECM proteins on the thin films, (d) cell handling and attachment to the AFM cantilever, and (e) measurement of adhesion forces. The protocol can be easily modified for other cell types and substrate proteins.
authors.affiliation: Leibniz Institute of Polymer Research Dresden, Institute for Biofunctional Polymer Materials, Dresden, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Friedrichs
authors.forename: Carsten
authors.initials: C
authors.lastname: Werner
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Müller
mesh_terms: D002448:Cell Adhesion / Q000235:genetics*; D003094:Collagen; D004338:Drug Combinations; D005109:Extracellular Matrix / Q000737:chemistry / Q000235:genetics; D005353:Fibronectins / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D007797:Laminin; D008299:Maleic Anhydrides / Q000737:chemistry; D008967:Molecular Biology / Q000379:methods*; D011108:Polymers / Q000737:chemistry; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D011509:Proteoglycans; D059010:Single-Cell Analysis; D013057:Spectrum Analysis
chemical_list: D004338:Drug Combinations; D005353:Fibronectins; D007797:Laminin; D008299:Maleic Anhydrides; D011108:Polymers; D011506:Proteins; D011509:Proteoglycans; C059485:matrigel; D003094:Collagen