IPSC-derived neurons were tested for calcium signaling dynamics using the FLIPRTETRA High Throughput Cellular Screening System as described in Vetter et al.35. Briefly, cells were loaded with 5 μM Fluo-4-AM in physiological salt solution (PSS). To allow for completion of de-esterification, cells were incubated for 10–15 minutes with PSS. After 2 washes with PSS, cells were transferred to the FLIPRTETRA (Molecular Devices, CA, USA) fluorescent plate reader and Ca2+ responses to activation with 50 mM KCl measured using a cooled CCD camera with excitation at 470–495 nm and emission at 515–575 nm. Camera gain and intensity were adjusted for each plate to yield a minimum of 1000 arbitrary fluorescence units (AFU) baseline fluorescence. Prior to addition of KCl, 10 baseline fluorescence readings were taken, followed by fluorescent readings every second for 300 seconds. Delta F/F was calculated for each time point as (fluorescence at time t – avg. baseline fluorescence)/(avg. baseline fluorescence) to give a normalized measure of fluorescence.
