H6147

Polyacrylamide gel substrates were prepared according to Wang and Pelham [71 (link)]. Briefly, No. 0 glass-bottom culture dishes (MatTek P35G-0-20-C; Ashland, MA, USA) were treated with 0.1 M NaOH, 97% (3-aminoproyl) trimethoxysilane (Sigma-Aldrich 281778; St. Louis, MO, USA), and 0.5% glutaraldehyde (Polysciences 01909; Warrington, PA, USA). Culture dishes were stored in a desiccator for up to 2 weeks until use. A 4.6 kPa hydrogel was made as follows: 100 µL of 40% acrylamide solution (Fisher Scientific BP1402; Waltham, MA, USA), 100 µL of 2% bis-acrylamide solution (Fisher Scientific BP1404; Waltham, MA, USA), 10 µL of 1 M 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES, Sigma-Aldrich H6147; St. Louis, MO, USA), 790 µL of deionized water, 6 µL of 1% ammonium persulfate (Bio-Rad 161-0700), and 4 µL of 0.4% (v/v) TEMED, (Fisher Scientific BP150; Waltham, MA, USA). Next, 4 µL of polymer solution was quickly pipetted onto the activated glass culture dishes and covered with a 12 mm No. 1.5 circular coverslip (Fisher Scientific 12-545-80; Waltham, MA, USA). Then, the coverslip was removed and washed three times with 50 nM HEPES. Finally, 1 mL of 200 mg/mL of type I collagen solution was added to the gel and the hydrogel was kept at 4 °C overnight.