The migration of Dlxi1/2b::eGFP+ cells was imaged for 8–12 hrs under environmentally controlled conditions (37°C, 5% CO2) in intact, fused hSS-hCS using a confocal microscope with a motorized stage (Leica SP8). Fused hSS-hCS were transferred to a well of a 96–well plate (glass-bottom plates, Corning) in 200 μl of NM. Spheroids were incubated in an environmentally controlled chamber for 30–60 min prior to imaging. During a given recording session, up to 8 fused hSS-hCS were imaged at a depth of 50–150 μm and at a rate of 15–20 min/frame. For pharmacological manipulation, cells were imaged for 12 hrs to record a baseline. Then, the media was carefully removed and new media with small molecules (AMD3100 at 100 nM; nimodipine at 5 μM; or roscovitine at 15 μM) was gently added to the well. The field of view was readjusted to capture the previous region of interest and cells in fused hSS-hCS were imaged for an additional 12 hrs.
