2.3. Cell–Cell Force Spectroscopy (CCFS)

CCFS experiments were carried out using a Nanowizard III system (Bruker, JPK Instruments, Berlin, Germany), coupled with an AxioObserver D1 (Zeiss, Oberkochen, Germany) inverted optical microscope. A CellHesion module (Bruker, JPK Instruments) was used to extend to 100 μm the vertical displacement range of the AFM. All experiments were carried out at 37 °C in PBS containing 2.0 mM CaCl₂ and 2.0 mM MgCl₂ and setting the force-curve length at 80 μm to achieve complete detachment of the cell probe from the target cell. For each set of experiments, only the measurement condition in consideration was changed while keeping the rest of the parameters unaltered. All the acquired F–D curves were processed with the JPK Data Processing software to correct for the bending of the cantilever and to remove the baseline offset and exported in txt format for further analysis.

Approach and retract speeds: the approach and retract speeds were also analyzed. For all the cases, the contact was kept for 45 s using the constant-height as the delay mode (see below). The setpoint force, hence the maximum force exerted between the two cells, was set at 1.0 nN. For the evaluation of the approach speed influence in the cell–cell interaction, the cantilever was lifted at a constant velocity of 10.0 μm/s. Different approach speeds (1, 2.5, 10, 25, and 50 μm/s) were investigated. To understand how the retract speed influences the measurements, the approach speed was set at a constant velocity of 10.0 μm/s. After the contact, the cell probe was lifted at 1, 2.5, 10, 25, and 50 μm/s. Over 10 F–D curves per speed were acquired for every experiment, for a total of 53 and 354 curves for approach and retract speeds, respectively.

Delay time: different extended pauses (1, 15, 30, 45, 60, and 120 s) were studied. The cell probe was lowered at a constant speed of 10.0 μm/s until the cell probe contacted the target cell and the preset force (setpoint force) of 1.0 nN was reached. The constant height was used as the delay mode. The cell probe was then retracted, lifting the cantilever at a constant velocity of 10.0 μm/s. A total of 211 curves were acquired.

Setpoint force: three setpoint forces (1, 10, and 30 nN) were studied. The cell probe was lowered at a constant speed of 10.0 μm/s until the cell probe contacted the target cell and the preset force was reached. The contact was kept for 45 s using the constant height as the delay mode. Then the cell probe was retracted, lifting the cantilever at a constant velocity of 10.0 μm/s to register the F–D curve. At least 33 F–D curves per each setpoint were acquired for a total of 100 F–D curves.

Delay mode: the contact between the two cells can be maintained in two different modalities, constant-force and constant-height. In constant-force, after reaching the setpoint force, the piezo actuator compensates for any change of the interaction force associated with the cell shape’s adaption and remodeling under an applied load. In constant height mode, the piezo actuator maintains a fixed position after reaching the setpoint force. In this second case, the cell position is fixed, but the interaction force can change due to the cell’s ability to remodel its shape. The cell probe was lowered and retracted at a constant speed of 10.0 μm/s. The cell probe was kept in contact with the target cell for 45 s after the preset force of 1.0 nN was reached. A total of 52 F–D curves for constant force mode and 41 F–D curves for the constant height mode were analyzed.