Single cell force spectroscopy (SCFS)

Prior to experiments, cells were cultured on poly-d-lysine functionalized glass in culture medium, in the CO₂ incubator for at least 2 h, then washed very carefully two times with PBS solution (pH 7.4). All SCFS measurements were carried out using standard atomic force microscopy (Nanowizard II, JPK) working in force spectroscopy mode. To prevent significant changes in morphology or biochemistry of living cells outside the incubator, each sample was measured within 3 h. During that time, at least two force maps (8 × 8 pixels grid, scan size of 8 × 8 µm²) in the center of the cells were recorded. The force set point was set at 0.5 nN and the approach and retract speeds were kept at 2 μm/s. For a given cell line type at least 25 living cells were measured. The cantilevers (MLCT-A, Nominal Constant 0.07 N/m, Bruker Corporation) were calibrated before the SCFS measurements both in air and in PBS solution using Thermal Noise method. The force–displacement curves between contact and 200 nm deformation depth have been analyzed, and the softer the sample the larger the deformation recorded. The evaluation of cells elastic properties, described quantitatively through the Young’s modulus, was obtained by force curves analysis with the Hertz–Sneddon contact mechanics for a paraboloidal tip^31,32.