Cell Stiffness Measurement using AFM

The stiffness of cells was measured using an atomic force microscope (Bruker NanoScope) coupled to a confocal microscope (TCS SP5II; Leica), as described previously^{50 (link),51 (link)}. The point-and-shoot procedure (NanoScope software; Bruker) was used to measure cell stiffness. All cells were kept in CO₂-independent cell culture medium during the measurement. A fluorescent 10 μm polystyrene bead (Invitrogen) was glued to silicon nitride cantilevers with nominal spring constants of 0.06 N m⁻¹ (NP-S type D; Bruker). Indentations were performed using the single force option with a total indentation depth of 50–100 nm. To obtain cell stiffness values from force curves, PUNIAS software was used as described previously^{50 (link),51 (link)}. Multiple force displacement curves (at five different locations) were fitted to the Hertz model to calculate cell cortical stiffness (Young’s modulus).