Jpk nanowizard 4 bioafm

We fixed the cell-cultured MPM onto the MPM holder dish using a perfusion system (custom-made by Nagata Industry Co.) with the membrane facing up. The MPM was bathed in Leibovitz’s L-15 medium (Thermo Fisher Scientific) supplemented with 1% penicillin/streptomycin (Fig. 1f). Then, the MPM holder dish was set on the stage of an inverted fluorescence microscope (Eclipse Ti2, Nikon) coupled to a JPK NanoWizard 4 BioAFM (Bruker). The temperature was kept at 37 °C using the heater equipped with this microscope to observe live cells. All AFM imaging was performed using BL-AC40TS-C2 cantilevers (Olympus, spring constant approximately 0.1 N/m). We used the QI settings with following parameters: topography Imaging: 2.5 × 2.5 or 0.5 × 0.5 μm scale, 64 × 64 pixels, Z-length 1 μm, setpoint 0.1 nN, speed 166 μm/s; imaging at the 100 × 100 nm scale: 64 × 64 pixels, Z-length 50 nm, setpoint 0.1 nN, speed 166 μm/s; for Young’s modulus measurement: 100 × 100 nm scale, 64 × 64 pixels, Z-length 2 μm, setpoint 0.1 nN, speed 166 μm/s; for the experiments of Fig. 4: 3.5 × 3.5 μm scale, 512 × 512 pixels, Z-length 1 μm, setpoint 0.1 nN, speed 166 μm/s.

Cell penetration measurements were performed in a JPK Nanowizard 4 BioAFM (Bruker Nano GmbH, Berlin, Germany). The nanoneedles were inserted into living cells at a speed of 10 µm/s, recording the cantilever deflection during the process until a specific set-point was reached, withdrawing subsequently the nanoprobe at the same speed. The penetration experiments were carried out in two different ways: (1) the nanoneedle above the cell was vertically moved down until a set point of 5 nN is reached, retracting afterwards (static mode); (2) similarly to the previous mode, the nanoneedle was vertically moved down, but in this case the cantilever was simultaneously vibrated with an oscillation amplitude of around 3 nm at its second resonance mode (40 kHz ~ 60 kHz), until the cantilever oscillation amplitude was decreased by 60%, retracting the nanoprobe after (dynamic mode). The number of penetration experiments was around 50 times per nanoprobe and method, always at some distance from the nucleus to avoid cell damage. Sensitivity and cantilever stiffness were calibrated using the thermal noise method as implemented in the JPK AFM control software. All the AFM experiments were performed at 37 °C in physiological conditions.