Glass or polystyrene culture dishes were coated with 10‐µg/ml fibronectin with or without the addition of 50‐µg/ml TTR fibrils or native tetramer in phosphate‐buffered saline (PBS) solution. Dishes were coated for 2 h in a 37°C, 5% CO2 incubator before cell plating.
Microgrooved substrates were prepared by molding 400 kPa polydimethylsiloxane (PDMS) from a parylene template to produce spaced grooves as done previously, with grooves 10‐µm wide, 5‐µm high, and ridges 10‐µm wide (Motlagh, Senyo, et al., 2003 (link)). Before coating with fibronectin or TTR fibrils, PDMS microgroove substrates were functionalized with 3‐aminopropyl triethoxysilane (Sigma‐Aldrich cat# 440140).
Polyacrylamide (PAA) substrates with a stiffness of 10 kPa were prepared as previously described (Li et al., 2016 (link)). Before polymerization, fluorescent microspheres (Invitrogen cat# F8807) were included in substrates for later traction force microscopy (Broughton et al., 2016 (link); Ribeiro et al., 2017 (link)). PAA substrates were functionalized with Sulfo‐Sanpah (Thermo Fisher cat# 22589) before coating with fibronectin or TTR fibrils.
Microgrooved substrates were prepared by molding 400 kPa polydimethylsiloxane (PDMS) from a parylene template to produce spaced grooves as done previously, with grooves 10‐µm wide, 5‐µm high, and ridges 10‐µm wide (Motlagh, Senyo, et al., 2003 (link)). Before coating with fibronectin or TTR fibrils, PDMS microgroove substrates were functionalized with 3‐aminopropyl triethoxysilane (Sigma‐Aldrich cat# 440140).
Polyacrylamide (PAA) substrates with a stiffness of 10 kPa were prepared as previously described (Li et al., 2016 (link)). Before polymerization, fluorescent microspheres (Invitrogen cat# F8807) were included in substrates for later traction force microscopy (Broughton et al., 2016 (link); Ribeiro et al., 2017 (link)). PAA substrates were functionalized with Sulfo‐Sanpah (Thermo Fisher cat# 22589) before coating with fibronectin or TTR fibrils.
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