For calcium imaging in monolayer, hCSs were dissociated and cultured for two weeks. Cells were loaded with 1 μM Fura-2 acetoxymethyl ester (Invitrogen) for 30 min at 37 °C in Neurobasal supplemented with B-27, washed with Tyrode’s solution (5 mM KCl, 129 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 30 mM glucose and 25 mM HEPES, pH 7.4) and placed in a perfusion chamber on the stage of an inverted fluorescence microscope (TE2000U; Nikon). Imaging was performed at room temperature (23–25 °C) on an epifluorescence microscope equipped with an excitation filter wheel and an automated stage. The Openlab software (PerkinElmer) was used to collect and quantify time-lapse excitation ratio images, and fluorescence images were analyzed with the IGOR Pro software (WaveMetrics). For calcium imaging in 3D cultures, intact hCSs were loaded with 1 μM Fluo-4 acetoxymethyl ester (Invitrogen) for 30 min at 37 °C in Neurobasal supplemented with B-27, washed and sliced in half. Live imaging was performed at the NMS (Stanford Neuroscience Microscopy Service, supported by a US National Institute of Health grant, NS069375) using a Zeiss LSM780 confocal microscope.
