Atomic Force Microscopy

Force mapping was performed in EBM-2 endothelial cells medium (Lonza, CC-3156) supplemented with EGM-2 Single Quots (Lonza, CC-4176) stabilized with 10 mM HEPES (Sigma, H3537) at 37°C using a BioScope Catalyst-AFM (Bruker Nano Surfaces, Santa Barbara, California, USA) in closed-loop mode with a ramp size of 2 µm, max. force of 1nN and a tip velocity of 2.6 µm/s. A Large-Radius-Bio-Probe (Bruker AFM Probes, Camarillo, CA, USA; 'The nature of this technology is the subject of a non-provisional patent application to Bruker Nano, Inc currently pending at the United States Patent and Trademark Office.') (tip radius 3.46 µm) was used. Spring constant of the cantilever (0.176 N/m) was determined with an interferometer (OFV-551, Polytec, Waldbronn, Germany). Deflection sensitivity was adjusted according to the SNAP procedure (Schillers et al., 2017). Each force map contained 16 × 16 force-distance cycles over an area of 150 × 150 µm. The analysis of the force–indentation curves was performed with PUNIAS software (http://punias.free.fr/) using the linearized Hertz model (Carl and Schillers, 2008).