Dafm 2x bioscope

For quantitative measurements of intracellular stiffness, cells were plated on fibronectin-coated low or high stiffness hydrogels in 10% FBS-containing DMEM (phenol red-free). AFM in force mode or force-volume mode was used to monitor the intracellular stiffness of single adherent cells on hydrogels using a DAFM-2X Bioscope (Veeco, Woodbury, NY) mounted on an Axiovert 100 microscope (Zeiss, Thornwood, NY). Cells were indented with a standard silicon nitride cantilever (spring constant =0.06 N/m) with a conical tip (40 nm in diameter). To quantify the cellular stiffness (E; Young’s modulus), the first 400 nm of tip deflection from the horizontal was fit with the Hertz model for a cone (68 ). For each experiment, 5–10 measurements of intracellular stiffness were collected near the periphery of each cell, and each experiment analyzed 8–10 cells per condition. AFM force curves were analyzed and converted to Young’s modulus using custom MATLAB scripts generously provided by Paul Janmey. Results from whole cell force-volume maps are representative of 7 (low stiffness) or 8 (high stiffness) cells.

AFM was conducted with a DAFM-2X Bioscope (Veeco, Woodbury, NY) mounted on an Axiovert 100 microscope (Zeiss, Thornwood, NY) using a triangular silicon nitride cantilevers with a conical tip (Veeco, model: DNP-10) for indentation over the cell lamella (as opposed to cell nucleus). The indentation was carried out at a 1 Hz loading rate and a ramp size of 3 μm. The spring constant of the cantilever, calibrated by resonance measurements, was typically 0.06 N/m. To quantify cellular stiffness, ~80 force–distance curves from 18 cells in four different samples were collected and analyzed according to the Hertz model modified for a conical probe.