Cellhesion module

We used a Nanowizard II AFM (JPK Instruments, Berlin, Germany) mounted on a Zeiss microscope (Carl Zeiss, Jena, Germany). This configuration allows to carry out AFM measurements and simultaneously observe the cells using phase contrast or fluorescence modes. This AFM is also equipped with the 'CellHesion' module (JPK Instruments, Berlin, Germany). This module enables a long-range vertical displacement of the stage up to 100 µm which makes force spectroscopy measurements possible including cell-cell interactions. In parallel, a vertical piezotranslator (PIFOC, Physik Instrumente, Karlsruhe, Germany) is mounted on the microscope objective to move the objective concurrently with the microscope stage and focus on cells while carrying out AFM measurements. All the measurements were carried out at 37°C using the Petri Dish Heater (JPK Instruments, Berlin, Germany).

Acquisition of adhesion data was performed using a NanoWizard II AFM (JPK Instruments, UK) employing a CellHesion module (JPK Instruments, UK), operating in force spectroscopy mode at 18°C. The sample and cantilever were immersed in acidic and alkaline aqueous solutions, contained within a clean glass Petri dish. The solutions were manufactured using dH₂O and pH adjusted with NaOH 0.01 M or HCl 0.01 M, to achieve solutions of pH 3, 5, 7 and 9. The samples were immobilised using a double-sided Shintron adhesive tape (Agar Scientific, UK).
The vertical deflection and z-axis displacement data were recorded at a frequency of 10 kHz. A grid of 100 force-displacement curves was acquired for each sample/liquid combination, equally spaced over an area of 100 μm × 100 μm. The force-displacement data were analysed using JPK Data Processing software (JPK Instruments, UK).
A schematic of the measured forces is shown in Fig. 1. The out-of-contact repulsion force (F_rep) was measured in the first peak from right to left of the approaching curve, the height of the triangle that was formed in the peak from the origin to the highest part of the peak until the signal was stable in the approach curve was measured as the force. The jump to force (F_JT) was measured in the minimum point of the approach curve, and the pull-off force (F_PO) was measured in the minimum point of the retract curve.

Cell surface stiffness was examined by indentation assay using an AFM cantilever as described previously^{55 (link), 56 (link)}. Briefly, force probe was prepared by attaching a glass bead (ca. 100 μm diameter) to a tipless silicon cantilever (450 μm long, 50 μm wide, 2 μm thick; nominal spring constant 0.02 N/m; TL-CONT, Nanosensors) using two component Araldite epoxy glue. The cells were set on a plastic culture dish, and pressed from the apical side with a force of 20 nN (the approach and retraction speeds were set to 1.0 μm/s) by the force probe. The measurement using a contact mode was carried out with the Nano-Wizard system with the Cell-Hesion module (JPK), and the data analysis was done with the JPK data processing software (JPK Instruments), where the cell elasticity (Young’s modulus) in the apical region was estimated from a force–distance curve using the Hertz model. The cells were cultured in Leibovitz’s L-15 medium (Thermo Fisher). Nocodazole treatments were given for 60 min at 37 °C before the measurements. All measurements were performed at room temperature.