Jpk data analysis software

AFM images were analyzed using JPK data analysis software (JPK Instruments, Berlin, Germany). The stiffness (Young’s modulus) of the collagen sheets was determined at each indentation position with a lab-made MATLAB application (Version R2018a, MathWorks, Inc., Natick, MA, USA) using the individually determined optical lever sensitivity and cantilever spring constant. The application automatically corrected for offset and tilt of the baseline and determined the contact point of each IT-AFM force curve [31 (link)]. Subsequently, a modified Hertz model for a four-sided pyramidal indenter, with a Poisson’s ratio of 0.5 and a tip half-opening angle to the edge of 17.5°, was fitted to the indentation part of the curves [29 (link),32 (link)]. The whole indentation curve, including the contact point but omitting the first 500 nm, was fitted to extract the Young’s modulus at each position on the sheets. Force curves and fits were manually inspected and discarded if the quality of the curve did not allow for a reliable fit. Stiffness (Young’s modulus) distributions were plotted, and Gaussian distributions were fitted to them using IGOR Pro 8.03 (WaveMetrics, Inc., Portland, OR, USA).

Cell–ECM adhesion forces were quantified by approaching cells to substrate-coated PDMS supports with 5 μm s^–1 until a contact force of 1 nN was reached. The cantilever was maintained at constant height for contact times of 5, 20, 60, 120, 240, or 360 s. The order of contact times was randomized for each cell to exclude memory effects of the cells on experimental sequences. Thereafter, the cantilever-bound cell was retracted from the substrate at 5 μm s^–1 for 100 μm until cell and substrate were fully separated. After the experimental cycle, cells were allowed to recover from adhesion measurement for the time of the contact time before measuring the adhesion force for a different contact time. The area on the substrate was altered after every adhesion force measurement cycle. Adhesion forces of cantilever bound cells were quantified for all contact times unless morphological changes, such as cell spreading or cell division, were detected. The order of contact times was randomized. Adhesion forces were determined after the retraction force-distance curves were drift- and baseline corrected using the JPK data analysis software (JPK Instruments). Adhesion force strengthening was determined as the slope of linear fits to all adhesion forces and contact times (PRISM).

JPK data analysis software was used to analyze force curves. We selected force curves for further analysis which had a contour length between 40–150 nm and would fit a worm-like chain (WLC). The force curves were smoothed, the x-axis leveled, both axes set to 0, and the force curve corrected for cantilever bending to obtain tip-sample separation, z. A WLC fit was obtained for each force curve while rupture force and contour length data were recorded. Data was sorted in order of rupture force and the highest rupture force data were eliminated using Poisson statistics to eliminate force curves that were likely the result of simultaneous multiple unbinding events [38 (link)].