Arrow tl1 cantilevers

Adhesion was characterised using single-cell force spectroscopy (SCFS), as described previously [44 (link), 45 (link)]. Tip-less Arrow TL1 cantilevers (Nanoworld AG, Switzerland) with a low spring constant were used (0.03 N/m). Cantilevers were sterilized with UV (10mins), before being functionalised in poly-L-lysine (25 μg/ml, 30mins, RT), and fibronectin (20 μg/ml, 2 h, 37 °C). A single cell was captured at the end of the cantilever with a set force (0.8-1 nN) and contact time (8-10s). After attachment, the cell was left to recover for > 5 min, allowing surface binding. The cantilever-attached cell was brought into contact with a substrate cell, until a 1nN contact force was reached. The two cells were attached for 10s to allow cell-cell adhesion, after which the cantilever was retracted at a constant speed (5 μm/sec). Force-displacement curves were measured until complete detachment (pulling length of 40-90 μm). Each procedure occurred in triplicate with 45 s intervals.

A total of 48 h prior to an atomic force microscopy (AFM) probing, cells were seeded into glass bottom dishes (WPI, Sarasota, FL, USA) and cultured as described above. For AFM indentation experiments, a Nanowizard 4 (JPK Instruments/Bruker, Berlin, Germany) was used. Arrow-TL1 cantilevers (Nanoworld, Neuchatel, Switzerland) with a nominal spring constant of 0.035–0.050 N/m that had been modified with a polystyrene bead of 5-µm diameter (Microparticles GmbH, Berlin, Germany) were calibrated by the thermal noise method, using built-in procedures of the AFM software. To probe a selected cell, the cantilever/bead was positioned over the nuclear region and three repeated indentation tests were performed using an approach/retraction speed of 5 µm/s and a relative set point of 2.5 nN. For morphotype classification, a phase-contrast image was recorded from each cell. All experiments were performed at 37 °C using a Petri dish heater (JPK instruments/Bruker) and in a CO₂-independent medium (Gibco Corp.). The resulting force distance curves were analyzed using the JPK data processing software (JPK instruments/Bruker). Force distance data were corrected for the tip sample separation and fitted with the Hertz/Sneddon model fit for a spherical indenter to extract the apparent Young’s modulus [25 (link),26 (link),27 (link)]. A Poisson ratio of 0.5 was assumed.

For AFM indentation experiments on single SGBS and 3T3-L1 cells (RRID:CVCL_0123), a Nanowizard I or IV were used (JPK Instruments/Bruker Berlin). Arrow-TL1 cantilevers (Nanoworld) with a nominal spring constant of 0.035 – 0.050 N/m that had been modified with a polystyrene bead of 5 µm diameter (microparticles GmbH), were calibrated by the thermal noise method using built-in procedures of the AFM software. Cells growing on glass slides were gently rinsed with CO₂-independent medium, which was also used for experiments. Then the cantilever was lowered with a speed of 5 µm/s onto the cells surface until a relative set point of 2.5 nN was reached and retracted. The resulting force distance curves were analysed using the JPK image processing software (JPK Instruments). Force-distance data were corrected for the tip-sample separation and fitted with the Hertz/Sneddon model fit for a spherical indenter to extract the apparent Young’s modulus^{61 ,62 (link)}. A Poisson ratio of 0.5 was assumed. All experiments were performed at 37 °C.