*4.12. Calcium Imaging*

For calcium imaging, NSCs were allowed to differentiate for 6 weeks (see NSC differentiation section) on Glass-Bottom P35 culture dishes coated with 15 μg/mL Poly-L-Ornithine (Sigma, St. Louis, MO, USA, P3655) at RT for 1 h and 10 μg/mL laminin at 37 ◦C for 2 h (Thermo Fisher Scientific, Waltham, MA, USA, 23017-015) [**?** ]. The day of the experiment, cells were loaded with 1 ng/μ<sup>L</sup> of Fluo-4 AM (Molecular Probes, Eugene, OR, USA, F14201) diluted in growth medium without Phenol Red and incubated at 37 ◦C and 5% CO2 for 30 min. Before imaging, medium was changed to fresh growth medium without Phenol Red to wash away excess Fluo-4 AM. Time-lapse recordings were acquired using a Zeiss confocal microscope at maximal speed during 30 min. At minute 6, 90 mM KCl was added to the culture plate. Four videos of each condition and independent culture plate were recorded for analysis. For quantification, 10 cells with response to KCl were chosen randomly, and the type of response was analyzed.
