F8800

After 16 h of culture on PA hydrogels with embedded fluorescent beads (F8800, Thermo Fisher Scientific), the cells were stained for CellTracker Deep Red dye (C34565, Thermo Fisher Scientific) according to the manufacturer's protocol. Imaging was done in FluoroBrite DMEM (A1896701, Thermo Fisher Scientific), 20% FBS (Gibco, 10437028), and 1% penicillin-streptomycin solution medium. Images of beads and cells were taken before and after the addition of 10% SDS (SDS001.500, Bioshop). Traction stress maps were obtained using ImageJ macros developed in prior published work (Martiel et al., 2015 (link); Tseng et al., 2012 (link)). Using MATLAB, cell masks were obtained by expanding the original CellTracker binarized image by 70 pixels in all directions. Only traction stress values inside the cell masks were considered.

PA hydrogels were fabricated according to the protocol described by Tse and Engler (2010) (link) with some modifications: hydrogels were cast on 35 mm glass bottom dishes (D35-14-1-U, Matsunami Glass, Osaka, Japan) instead of coverslips, and deposition of NaOH was followed by immediate aspiration instead of heated evaporation. Hydrogels were then tethered with either fibronectin (FN; fibronectin bovine plasma, F1141, Sigma, St Louis, MI, USA), collagen (COL; collagen I, rat tail, A1048301, Gibco, Waltham, MA, USA) or laminin (LAM; 11243217001, Roche, Basel, Switzerland) at a 100 mg/ml concentration according the protocol reported by Tse and Engler (2010) (link). Briefly, polymerized hydrogels were covered with sulfo-SANPAH (22589, Thermo Fisher Scientific, Waltham, MA, USA), exposed to UV light for 15 min, and then washed with HEPES buffer three times for 15 min. The hydrogels are then incubated in the ECM proteins overnight at 37°C. Young's moduli of the hydrogel formulations were measured by compression testing using a Mach-1 micromechanical system (Biomomentum, Laval, Quebec, Canada) controlled by a Universal Motion Controller (Newport, Irvine, CA, USA) (Fig. S1A).
For traction force microscopy, PA hydrogels were fabricated following the same protocol above with the addition of embedding 0.1 µm diameter fluorescent beads (F8800, Thermo Fisher Scientific) at a 1:200 dilution.