For sorting of dissociated two-month-old hiPSC neurons, cultures were dissociated in trypsin for 5 minutes, washed in DMEM, centrifuged at 500×g and resuspended in PBS. Cells were fixed in 4% paraformaldehyde in PBS at 4°C for 10 minutes. Cells were washed in PBS and aliquoted into 96-well conical plates. Cells were blocked in 5% donkey serum with 0.1% saponin at room temperature for 30 minutes. The following primary antibodies and dilutions were used for one hour at room temperature: rabbit anti-βIII-tubulin (Sigma), 1:200; mouse anti-βIII-tubulin (Covance), 1:200; rabbit anti-GAD56/67 (Sigma), 1:200. Cells were washed and then incubated with secondary antibodies at 1:200 for 30 minutes at room temperature: Alexa donkey 647 anti-rabbit (Invitrogen), and Alexa donkey 488 anti-mouse (Invitrogen). Cells were washed three times in PBS and stained with 0.5µg/ml DAPI (4',6-diamidino-2-phenylindole). Cells were resuspended in PBS with 5% donkey serum and 0.1% detergent saponin. The homogeneous solution was filtered through a 250-µM nylon sieve and run in a BD FACS Caliber. Data were analyzed using FloJo.
