35 mm glass bottomed dish

E10.5 spinal cord slices for live timelapse microscopy were placed on a 12 mm Millicell cell culture insert (MerckMillipore) in a 35 mm glass-bottomed dish (Greiner BioOne) incubated at 37 °C and 5% CO₂. The legs of the cell culture insert were sanded down to decrease the distance from the glass to the tissue. Primary neural stem cells dissociated from E10.5–E11.5 Venus::HES5 spinal cords were maintained as a line for up to 10 passages and plated in 35 mm glass-bottomed dish (Greiner BioOne) for live imaging. 1.5 mls of DMEM F-12 (Thermo Fisher Scientific) media containing 4.5 mg/ml glucose, 1× MEM non-essential amino acids (Thermo Fisher Scientific), 120 μg/ml Bovine Album Fraction V (Thermo Fisher Scientific), 55 μM 2-mercaptoethanol, 1x GlutaMAX (Thermo Fisher Scientific), 0.5× B27 and 0.5× N2 was added. Movies were acquired using Zeiss LSM880 microscope and GaAsP detectors. For slice imaging a Plan-Apochromat ×20 0.8 NA objective with a pinhole of 5 AU was used. Ten z-sections with 7.5 μm interval were acquired every 15 min for 18 h. DMSO (Sigma) or 2 μM DBZ (Tocris) was added to media immediately before imaging. For imaging dissociated cells a Fluar ×40 1.3 NA objective with a pinhole of 6.5 AU was used. Six z-sections with 3.7 μm interval were acquired every 10 min for 24–48 h.