Petri dish heating stage

Live cell measurements were performed using a Bruker BioScope Catalyst AFM system (Bruker, Santa Barbara, CA) mounted on an inverted Axiovert 200M microscope system (Carl Zeiss, Göttingen, Germany) equipped with a Confocal Laser Scanning Microscope 510 META (LSM 510 Meta, Carl Zeiss) and a 40x (0.95 NA, Plan-Apochromat) objective lens (Carl Zeiss). A Petri dish heating stage (Bruker) was used to maintain physiological temperature (37 °C) of cells during measurements. Modified AFM microcantilevers with an attached 25 µm-diameter polystyrene microsphere were obtained from Novascan (Novascan, Ames, IA). The AFM probe spring constant was obtained using the thermal tune method built into the AFM system. Calibrated spring constants for the cantilevers ranged from 0.5 N/m to 1 N/m. After cantilever calibration, the AFM probe was placed on top of the nuclear region of an adherent cell. The deflection setpoint was set between 20 nm and 25 nm, yielding applied forces between 5 nN and 18 nN. The force curve ramp rate was set to 0.5 Hz and the probe speed ranged between 1.9 µm/s and 2.4 µm/s. Multiple consecutive quasi-static force curves were collected on each individual cell with a deflection trigger of 25 nm.