hMGEOs and hCOs derived from H1 hESCs and hiPSC (1090) were used for calcium imaging, respectively. Organoids were transduced with AAV1.syn.GCaMP6s.WPRE.SV40 (Penn Vector Core) and 10∼15 days after transduction intact organoids were used for calcium imaging. Nikon inverted microscope (Eclipse TS100) was used to observe calcium surges with 10× (area-scale) or 20× (single cell level) objectives at 488-nm excitation. Time lapse images were captured using a Digital CCD camera (QICAM: FAST 1394) and Qcapture Pro7 software (QICAM) at a speed of 1 frame/sec (FPS). For chemical treatment, images were obtained in the same location before and 10 min after adding either TTX (1 μM) or bicuculline (50 μM). Tracings of single cell calcium surges were analyzed using Matlab software with FluoroSNNAP code (Patel et al., 2015). Area-scale image series were analyzed using Fiji software (Schindelin et al., 2012).
