Preparation and synthesis of polyacrylamide hydrogels

To prepare glass surfaces for gel binding, 3 µl bind-silane (GE Healthcare), 47 µl glacial acetic acid and 950 µl ethanol (Fisher Scientific) were mixed together, and 200 µl of this mix was pipetted onto glass-bottomed dishes (No. 0, 14 mm diameter; MatTek). After 3 min, dishes were rinsed with ethanol and left to dry at room temperature. Separately, to clean glass coverslips (No. 0, 13 mm diameter), they were initially sonicated in acetone for 15 min, rinsed twice with water and rocked for 1 h in NaOH (1 M). Coverslips were washed extensively with water and stored in 70% ethanol until use.

Hydrogels were synthesised using ProtoGel (National Diagnostics) adapted from a method described by Wang and Pelham (1998). ProtoGel, a stabilised mixture of 30/0.8% (w/v) acrylamide/bis-acrylamide solution was mixed with PBS (90, 80 and 50% of the final volume) to synthesise gels with Young's moduli of 1, 22 and 142 kPa, respectively. The ProtoGel mixture was degassed in a vacuum chamber whereupon 100 mg/ml ammonium persulfate (Sigma-Aldrich; 1/100) and N,N,N′,N′-tetramethylethane-1,2-diamine (TEMED; Sigma-Aldrich; 1/1000) were added to the solution. The solution was added onto a silianised glass bottom dish and a clean glass coverslip was immediately placed on top. Post-gelation, the dish was flushed with PBS and the top coverslip was removed with forceps. The gels were washed twice with PBS to remove unbound acrylamide and stored at 4°C.