F8810

For traction force experiments, MSCs from each of the three substrates were trypsinized and re-seeded onto fluorescent microsphere (0.2-μm-diameter, F8810, Invitrogen, Carlsbad, CA)-embedded polyacrylamide (PA) hydrogels (Young’s Modulus, E = 10 kPa, 3000 cells/cm², prepared on glass slides). Cells were allowed to attach to the hydrogels for ~18 hrs. Cells and embedded beads were imaged using a Deltavision Deconvolution Microscope in an environmental chamber (37°C, 5% CO₂). TFM data analysis (stack alignment, PIV, and FTTC) was carried out using an FTTC plug in in Image J and a custom MATLAB script as in ^{26 (link),27 (link)}.

Traction force microscopy was performed as described previously ^{(23 (link), 38 (link))} Briefly, prior to polymerization, polyacrylamide (PA) hydrogels were UV-cured to a modulus of 10kPa (as verified by AFM). Hydrogels were subsequently washed three times with PBS and incubated with fibronectin solution (20 μg/ml, F1141, Sigma Aldrich) for 1 hour. MEFs were seeded on PA hydrogels at 1000 cells/cm² and cultured for 18 hours in DMEM/10% FCS before carrying out traction force microscopy (TFM). Cells (phase) and embedded beads (0.2-μ-diameter fluorescent microspheres, F8810, Invitrogen, Carlsbad, CA) were imaged using ZEISS Axio AXIO Observer fluorescence microscope at 40X magnification. Image sequences for each cell were taken before and after cell lysis with lysis buffer (10% Sodium dodecyl sulfate and Triton X-100 at 1:100).
TFM data analysis was performed using a Fourier transform traction cytometry plugin in Image J and a custom MATLAB (The MathWorks) as in ^{(23 (link), 38 (link))}. In some studies, cells were treated with 10μM Fasudil (Sigma Aldrich, catalog CDS021620), a potent RhoA inhibitor ^{(48 (link))}, for 30 min before measurement of traction forces.