Primate EB FACS Isolation Protocol

FACS was performed as previously described with some minor modifications (Kobayashi et al, 2017 (link)). Briefly, primate EBs were harvested on day 4, washed with PBS, and incubated in 0.25% trypsin/EDTA in a thermomixer at 850 rpm at 37°C for 9–13 min. The reaction was stopped with 3% FBS in PBS, and the cell suspension was pipetted until it was dissociated into single cells and then passed through the strainer. The cells were centrifuged, and the cell pellet was resuspended in 3% FBS in PBS. Then, the dissociated cells were stained for CXCR4-APC (306510; BioLegend) and INTα6-BV421 (313624; BioLegend) for 1 h in the dark on ice. Afterwards, the cells were washed in 3% FBS in PBS and subjected to FACS using SONY Cell Sorter SH800Z.