Traction Force Microscopy on Polyacrylamide Gels

Polyacrylamide gels (0.69 and 4.07 kPa shear modulus, 40 μm thickness) were prepared on glass coverslips with embedded 40 nm fluorescent beads (Thermo Scientific, TransFluoSpheres 633/720) and the surfaces were coated with neutravidin as described^{71 (link)}. TGT was immobilized on the surface and the Cy3 signal of TGT was checked to confirm the immobilization. cRGDfK or MUPA-LDVPAAK ligand was conjugated on the immobilization strand of TGT to prevent force-induced rupture. Cells were seeded on the gels for 1 h. The bead images were taken before and after cell removal with the addition of 0.1% SDS. Bead displacements were determined using particle image velocimetry, and the corresponding contractile energy was estimated with Fourier transform traction cytometry, using ImageJ plugins^{72 (link)}. The cell area was determined by manually drawing an ROI in the DIC channel. Imaging was performed on Nikon Eclipse Ti2 microscopes equipped with Perfect Focus, CSU-W1 spinning disk, and Hamamatsu Orca-flash 4.0 v3 camera. Illumination was provided by optical fiber (Oz Optics) coupled solid-state lasers: 561 nm (200 mW) for Cy3 and, 655 nm (100 mW) for fluorescent beads from Spectral Applied Research. Emission was collected via single-bandpass emission filter (605/52 nm) and a long-pass LP647 nm filter for far-red from Chroma.