Calcium imaging analysis was performed on 2.5- to 3-month-old hiPSC neurons cocultured with wildtype human astrocytes (Sciencell) on acid-etched glass. Culture medium was removed and hiPSC cultures were incubated with 0.4 µM Fluo-4AM (Molecular Probes) and 0.02% Pluronic F-127 detergent in Krebs Hepes Buffer (KHB) (10 mM HEPES, 4.2 mM NaHCO3, 10 mM dextrose, 1.18 mM MgSO4•2H2O, 1.18 mM KH2PO4, 4.69 mM KCl, 118 mM NaCl, 1.29 mM CaCl2; pH 7.3) for one hour at room temperature. Cells were washed with KHB buffer, incubated for two minutes with Hoechst dye diluted 1:1000 in KHB, and allowed to incubate for an additional 15 minutes in KHB to equilibrate intracellular dye concentration. Time-lapse image sequences (100x magnification) were acquired at 28 Hz using a Hamamatsu ORCA-ER digital camera with a 488 nm (FITC) filter on an Olympus IZ81 inverted fluorescence confocal microscope. Images were acquired with MetaMorph.
