The preparation of micropatterned substrates for neuronal cell culture took place in an aseptic environment and all reagents used were sterile. To sterilize micropatterned substrates, they were rinsed with 100% ethanol and placed in a petri dish containing enough 100% ethanol to fully submerge them for 15 minutes. Substrates were removed from the petri dish using a sterilized fine forceps and completely air dried before placing them into a 24-well plate (about 15 minutes). For live cell imaging micropatterned substrates, which were glued to drilled dishes (see below), ethanol rinsing and drying was done in the same manner. When completely dried, substrates were coated with laminin (5ug/ml, Roche, # 11243217001) in PBS and incubated at 37°C in a humidified incubator for 1 hour. Before seeding cells onto substrates, laminin was aspirated off and substrates were washed once with PBS. For live cell imaging studies, an 18mm whole was drilled into 35mm plastic dishes. Micropatterned substrates were glued onto a drilled dish using Sylgard (Electron Microscopy Sciences, #21236) adhesive, which was cured briefly at 80°C on a temperature-controlled hotplate.
