Jpk nanowizard 3 system

For AFM measurements, a JPK Nanowizard III system (Bruker, Berlin, Germany) was used to perform imaging, force spectroscopy, and creep measurement. The AFM system was built onto an inverse optical microscope (Axio Observer Z1, Zeiss, Berlin, Germany), and a temperature-controlled liquid measurement chamber was used. Triangular MSCT cantilevers E with pyramidal tips (a nominal tip radius of 10 nm, resonance frequency of 38 kHz in the air, and a spring constant of 0.1 N/m; Bruker, Germany) were used. Prior to and after measurements, they were cleaned with EtOH, acetone, and 30 min periods of UV/O cleaning. Sensitivity and the spring constant were calibrated by the thermal tune method, making use of the equipartition theorem. The system was left to equilibrate 30 min prior to measurements. Each sample was produced in triplicates and measured for a maximum of three hours.

For AFM imaging, cells were seeded onto plasma-treated glass slides and fixed as described above. A JPK Nanowizard III system (Bruker, Germany) was used to perform imaging in contact mode. Triangular MLCT cantilevers (Bruker, Germany) with nominal spring constants of 0.01 and 0.02 N/m, a resonance frequency of 7 kHz in air and a tip radius of 20 nm were used. Spring constants of the cantilevers were calibrated using the thermal noise method [37 (link)]. Imaging was performed with force set points between 0.4 and 0.8 nN, line rates of 0.5–0.8 Hz, scan sizes ranging from 75 µm × 75 µm to 100 µm × 100 µm and a resolution of 512 × 512 pixels. Those measurements were performed in PBS at room temperature. Images were analyzed using JPKSPM software (Version 6.1.159). First, a polynomial surface was subtracted from the image and then a low-pass filter was applied. The height of at least 25 cells was determined from the AFM images.