title: Assay for characterizing the recovery of vertebrate cells for adhesion measurements by single-cell force spectroscopy.
abstract: Single-cell force spectroscopy (SCFS) is becoming a widely used method to quantify the adhesion of a living cell to a substrate, another cell or tissue. The high sensitivity of SCFS permits determining the contributions of individual cell adhesion molecules (CAMs) to the adhesion force of an entire cell. However, to prepare adherent cells for SCFS, they must first be detached from tissue-culture flasks or plates. EDTA and trypsin are often applied for this purpose. Because cellular properties can be affected by this treatment, cells need to recover before being further characterized by SCFS. Here we introduce atomic force microscopy (AFM)-based SCFS to measure the mechanical and adhesive properties of HeLa cells and mouse embryonic kidney fibroblasts while they are recovering after detachment from tissue-culture. We find that mechanical and adhesive properties of both cell lines recover quickly (<10 min) after detachment using EDTA, while trypsin-detached fibroblasts require >60 min to fully recover. Our assay introduced to characterize the recovery of mammalian cells after detachment can in future be used to estimate the recovery behavior of other adherent cell types.
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, 4058 Basel, Switzerland.
authors.forename: Rajib
authors.initials: R
authors.lastname: Schubert
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, 4058 Basel, Switzerland.
authors.forename: Nico
authors.initials: N
authors.lastname: Strohmeyer
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, 4058 Basel, Switzerland.
authors.forename: Mitasha
authors.initials: M
authors.lastname: Bharadwaj
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, 4058 Basel, Switzerland.
authors.forename: Subramanian P
authors.initials: SP
authors.lastname: Ramanathan
authors.affiliation: Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA 94305, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Krieg
authors.affiliation: Leibniz Institute of Polymer Research Dresden, Institute for Biofunctional Polymer Materials, Hohe Str. 6, 01069 Dresden, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Friedrichs
authors.affiliation: Karlsruhe Institute of Technology (KIT), DFG-Center for Functional Nanostructures, Wolfgang-Gaede-Str. 1a, 76131 Karlsruhe, Germany.
authors.forename: Clemens M
authors.initials: CM
authors.lastname: Franz
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, 4058 Basel, Switzerland. Electronic address: daniel.mueller@bsse.ethz.ch.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Muller
mesh_terms: D000205:Actomyosin / Q000378:metabolism; D000818:Animals; D001696:Biomechanical Phenomena; D002448:Cell Adhesion; D003599:Cytoskeleton / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D055595:Mechanical Phenomena; D051379:Mice; D018625:Microscopy, Atomic Force / Q000379:methods*; D021381:Protein Transport; D013997:Time Factors
chemical_list: D000205:Actomyosin
keywords: Atomic force microscopy; Collagen I; Ethylenediaminetetraacetic acid; Extracellular matrix; Fibronectin; Trypsin