Live imaging dishes

Cryo-preserved, predifferentiated iNeurons (i³Neurons or Piggybac-delivered NGN2 neurons) were thawed and plated on live-imaging dishes (MatTek) coated with poly-L-ornithine at a density of 300,000 neurons per dish. For each experimental condition, cells from at least two different batches of induction were used over three or more independent experimental cultures. iPSC-derived neurons were cultured in BrainPhys Neuronal Media (StemCell) supplemented with 2% B-27 (Gibco), 10 ng/ml BDNF (PeproTech), 10 ng/ml NT-3 (PeproTech), and 1 μg/ml laminin (Corning). 40% of the media was replaced with fresh media twice per week. For Piggybac-delivered NGN2 neurons, 10 μM 5-Fluoro-2′-deoxyuridine and 10 μM uridine were included at the time of plating to prevent survival of mitotic cells. These drugs were removed 24 h after plating. Live imaging experiments were performed 21 days after thawing predifferentiated iPSC-derived neurons (DIV21). On DIV18, iPSC-derived neurons were transfected with Lipofectamine Stem (Thermo Fisher Scientific) and 1–2.5 μg total plasmid DNA. Immunostaining experiments were performed at DIV14. The published protocol can be found on Protocols.io (https://doi.org/10.17504/protocols.io.x54v9dj4zg3e/v1).

Cryo-preserved, pre-differentiated iNeurons (i³Neurons or Piggybac-delivered NGN2 neurons) were thawed and plated on live-imaging dishes (MatTek) coated with poly-L-ornithine at a density of 300,000 neurons per dish. For each experimental condition, cells from at least two different batches of induction were used over three or more independent experimental cultures. iPSC-derived neurons were cultured in BrainPhys Neuronal Media (StemCell) supplemented with 2% B-27 (GIBCO), 10 ng/mL BDNF (PeproTech), 10 ng/mL NT-3 (PeproTech), and 1 μg/mL laminin (Corning). 40% of the media was replaced with fresh media twice per week. For Piggybac-delivered NGN2 neurons, 10 μM 5-Fluoro-2′-deoxyuridine and 10 μM uridine were included at the time of plating to prevent survival of mitotic cells. These drugs were removed 24 hours after plating. Live imaging experiments were performed 21 days after thawing pre-differentiated iPSC-derived neurons (DIV21). On DIV18, iPSC-derived neurons were transfected with Lipofectamine Stem (Thermo Fisher) and 1-2.5 μg total plasmid DNA. Somal pT73 RAB10 immunostaining (Figure S2C) was performed at DIV7. Published protocol can be found on Protocols.io (https://doi.org/10.17504/protocols.io.x54v9dj4zg3e/v1).