Bl ac40ts c2

In this study, atomic force microscopy (AFM) was performed using a NanoWizard 3 AFM system (JPK Instruments AG, Berlin, Germany) with a cantilever and a tetrahedral type probe (BL-AC40TS-C2; Olympus, Tokyo, Japan). AFM was used to measure the mechanical properties of the cells. The plot of the measured force against the deformation of the sample (force-distance curve) provides information on the material properties, such as Young’s modulus. Luo et al. reported that the dissolution of the actin cytoskeleton led to a significant decrease in Young’s modulus. Therefore, in this study, Young’s modulus was used as an index of cell stiffness [21 (link)].
The cells were grown on glass bottomed culture dishes at a density of 3.0 × 10⁴ cells/mL, incubated at 37 °C for 48 h. Thereafter, measurements were conducted in cell culture media at room temperature. All force curves and scanning field images (10 µm × 10 µm) were recorded at a resolution of 128 × 128 pixels in Quantitative imaging (QI) mode at 37 °C.
Measurement of cell stiffness using AFM was performed as previously described [22 (link)]. The data were processed by curve-fitting with the Hertz contact model using JPK data processing software. The geometric mean of Young’s modulus was calculated from the acquired Young’s modulus at each point of the cell for a given condition.

12 mm diameter coverslips were coated nonspecifically with 1 mg/ml collagen I solution at 4 °C overnight and then washed extensively with PBS. 5000 cells were then seeded onto each coverslip and allowed to attach overnight at 37 °C with 5% CO₂. Cells were then washed with PBS.
A 5 μm spherical silica microsphere (polysciences) was mounted to the tip of a PNP-TR-TL cantilever (NanoWorld) with UV-curable glue. The tip was then loaded onto a liquid perfusion cantilever holder. The cell samples with PBS were loaded onto the 37 °C environmental chamber on a Cypher ES AFM (Asylum Research). Contact mode was used to obtain the force curves. To confirm cell attachment and compatibility with the system, a different tip, BL-AC40TS-C2 (Olympus), was used to image the cells. GetReal™ was used to calibrate the tip spring constant and the inverse of the optical lever sensitivity (InvOLS), using the thermal method. The relative trigger point voltage was set to be 0.1 V, and the indentation depth was set to be 200 nm. 5–10 force curves per cell were obtained on cell periphery. All measurements were completed within 30 min after the placement of the sample. The Hertz model $E=\frac{3\left(1,-,{\mathrm{\nu }}^2\right)F}{4\sqrt{R{I}^3}}$ where E, elastic modulus; F, force; R, sphere radius; I, indentation, and ν, Poisson ratio of cell, was fitted to the approaching force curve to extract the apparent elastic modulus.