Acrylamide

Polyacrylamide substrates were synthesized for cell analysis as previously described[17 ]. Two different rigidity substrates were fabricated: soft with 8% acrylamide (Sigma Aldrich) and 0.02% bis-acrylamide (~2 kPa, GE Healthcare Life Sciences) and hard (8% acrylamide and 0.06% bis-acrylamide, ~8 kPa). Briefly, glass coverslips were activated by incubation in 1% 3-aminopropyltriethoxysilane followed by 0.5% glutaraldehyde. acrylamide solutions were made with 200 nm red particles (Invitrogen) and ammonium persulfate and tetramethylethylenediamine were added to initiate gel polymerization. This solution was then added to glass slides and an activated coverslip added on top. In order to allow for cell adherence, the surface was activated with 0.2 mg/mL sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)-hexanoate (G-Biosciences) and coated with 0.2 mg/mL rat tail collagen I (Corning), which has previously been shown to play a critical role in MSC differentiation [18 (link)], overnight at 4°C with agitation. Substrates were sterilized under UV light and incubated in media for at least an hour before use. Mechanical properties of the substrates were assessed via compression testing using a Bose Endura TEC ELF 3200 Uniaxial Testing System (data not shown). The Young’s modulus was calculated from the slope of the linear region of the stress vs. strain curve at less than 10% strain.