Epha4 fc

LN was used as a chemoattractive cue and EphA4-Fc as a chemorepulsive cue to create on/off biochemically patterned substrates (Tuft et al., 2018 (link)). We used the same micropatterned MA platforms used in neurite guidance studies as stamps to print LN or EphA4-Fc peptides on MA substrates lacking topographic patterns (Fig. 1) (Tuft et al., 2013 (link)). Micropatterned MA stamps were immersed in a solution of either LN (20 μg/ml, Sigma-Aldrich Cat#: L2020, Lot number: 121M4043V) or EphA4-Fc (10 μg/ml, R&D Systems). The peptide-„inked‟ stamps were pressed onto a smooth polymer. This method allowed us to establish precise patterns of peptides. We also formed peptide printed topographical materials by applying proteins to a stamp with an unpatterned surface and then printing them onto the ridges of a micropattern, thereby producing substrates with both topographic and biochemical micropatterns (Fig. 1). To assess the contribution of LN working synergistically with topographical grooves, we formed materials with LN adsorbed only in the grooves. This was done by placing sufficient LN solution to fill grooves and wicking away excess protein solution that was absorbed on the ridges by placing absorbent paper and a glass coverslip on the ridges, leaving the LN solution only in the substrate grooves.

Following the generation of unpatterned or micropatterned methacrylate thin-films, adsorbed biochemical borders were created on the surface to investigate their role in neurite pathfinding on physical guidance cues. To create biochemical borders, equal volumes of solutions with different bioactive species, e.g. one solution containing laminin (20 µg/ml, Sigma-Aldrich) and the other containing tenascin-C (TnC, 100 µg/ml, Millipore) or EphA4-Fc (10 µg/ml, R&D Systems), were separately deposited on a methacrylate polymer surface overnight at 4 °C. A glass coverslip for each solution was placed on the dispensed volume so as to cover half of the polymer film while aligning adjacent to the other coverslip. Protein solutions subsequently spread across the surface due to capillary forces between the glass coverslip and the methacrylate thin-film forming a boundary between the two proteins where the two cover slips join. Following adsorption, polymer films were rinsed three times with phosphate-buffered saline (PBS).