Polyacrylamide Gel Synthesis for Cell Culture

PAA gels with Young modulus E = 30 kPa—which matches approximately the stiffness of the majority of abdominal organs and the skin (Guimarães et al., 2020 (link))—were produced according to a previously published protocol (Bazellières et al., 2015 (link)). A PBS solution containing 12% acrylamide (161-0140; Bio-Rad), 0.15% bis-acrylamide (161-0142; Bio-Rad), plus 0.05% ammonium persulfate (A3678; Sigma-Aldrich) and 0.05% tetramethyl ethylenediamine (T9281-25 Ml; Sigma-Aldrich) was prepared and allowed to polymerize between a coverslip and a glass-bottomed dish (Mattek). Alternatively, 3% acrylamide and 0.03% bis-acrylamide were used to make 300 Pa gels. The PAA gel surface was then incubated with a solution of 2 mg ml⁻¹ Sulfo-SANPAH (4822589; Thermo Fisher Scientific) under ultraviolet light for 5 min (wavelength of 365 nm at 5 cm distance). After that, the excess Sulfo-SANPAH was removed by three consecutive 3-min washes with PBS. A solution of fibronectin 0.1 mg ml⁻¹ (F0895; Sigma-Aldrich) was added on top of the gels and left overnight at 4°C.