Gel functionalization with fibronectin

PA gels were functionalized with fibronectin using the cross-linker 6-((acryloyl)amino)hexanoic acid succinimidyl ester (Acryloyl-X SE; Thermo Fisher Scientific). The PA gel functionalization was adapted from a previous protocol (48). Briefly, desired amounts of 0.2% bis-acrylamide, 3% Irgacure 2959, 0.5 M Hepes (pH 6.0), and 0.3% Acryloyl-X SE were mixed and applied onto gels. Gels were overlaid with Sigmacote-treated coverslips and then placed under a UV source to activate photoinitiator for 10 min. The top coverslips were removed, and the gels were gently washed with 50 mM Hepes buffer (pH 6) for 5 min on ice three times. The gel substrates were then incubated in fibronectin (20 μg/ml) in 50 mM Hepes buffer (pH 8) overnight at 4°C. The PA gels were washed with phosphate-buffered saline (PBS). For silicone gel functionalization, the cured silicone gels were incubated in fibronectin (10 μg/ml) in PBS overnight at 4°C to allow fibronectin adsorption onto the gels and then washed with PBS.