3 aminopropylsilyl

To measure cell traction forces exerted by the cell, 5 kPa polyacrylamide (PAA) hydrogel substrates were prepared as described previously ^{[44 ,45 ]}. Briefly, 8% acrylamide and 0.1% bis-acrylamide (Bio-Rad), 2% N-Hydroxysuccinimide (dissolved in DMSO) (Sigma), 0.375 % 3-aminopropylsilyl (Thermo Fisher Scientific), 0.125% tetramethylethylenediamine (Millipore Sigma) were added in water to prepare the gels. In addition, 1% of 200 nm fluorescently labeled green beads (2% solid, Thermo Fisher Scientific) was added before gelation and the solution was left for 30 minutes to 1 h for polymerization at room temperature (RT). Sulfo-SANPAH was used for gel surface activation to facilitate protein conjugation for 3 – 5 min under UV illumination. Gels were laminated with rat tail collagen I at a concentration of 100 μg/mL. After 24 h of seeding, phase images, and fluorescence images of the bead were acquired before and after removal of the cells to relieve traction stress. For the TFM analysis, a custom-built Matlab code was used. The details of the calculation can be found in refs ^{[44 ,46 (link)]}. Briefly, the displacement field is calculated from stressed and relaxed images of the beads. A 32×32 pixel ROI is used for the image correlation calculation. By constrained Fourier transform traction microscopy (FTTM) the cell traction field is obtained from the displacement field.

Hydrogels are prepared as described in the previous work[35 (link)]. Briefly, for elastic substrate 8% acrylamide and 0.1% bis-acrylamide (Bio-Rad), 2% N-hydroxysuccinimide (dissolved in DMSO) (Sigma), 0.375 % 3-Aminopropylsilyl (Thermo Fisher Scientific), 0.125% tetramethylethylenediamine (Millipore Sigma) added into water to prepare elastic gel of stiffness 5kPa (G’=5kPa and G” =0Pa) and for viscoelastic gel (G’ = 5kPa, G” =600Pa) 14.3 % linear acrylamide (5%stock) and 0.15% bis-acrylamide (instead 0.1%) added to the same protocol as elastic substrate recipe (Fig 1(a)). Gels are laminated with (100 μg/ml) collagen type I (Corning) by incubation overnight at 4 °C.