Calcium Imaging of Neuronal Differentiation

For calcium imaging, NPCs were incubated for 56 days in 3 N medium^{20 (link)} for cortical-like differentiation. For further 14 days of differentiation, media were exchanged for BrainPhys^TM Neuronal Medium (STEMCELL Technologies Germany GmbH, Köln, Germany) plus supplements (20× NeuroCult SM1 Supplement, 10× N2 Supplement, 100 µg/ml BDNF, 100 µg/ml GDNF, 100 mg/ml cAMP, and 50 mg/ml L-ascorbic acid). For visualization of calcium signals, neurons were stained with 1 µM Cal520^® AM (AAT Bioquest^®, CA, USA) for 30 min and imaged for 10 min at a rate of 20 frames per second using spinning disc confocal microscopy (Cell Observer SD, Carl Zeiss Microscopy GmbH, Oberkochen, Deutschland equipped with an an iXon DV885 EMCCD camera, Andor Technology, Belfast, UK).