To quantitatively analyze the phenotype of generated cells, we performed FACS analysis of stained cells at day 21 of differentiation. Differentiated cells were trypsinized and fixed in Fix/Perm solution for 30 min, and incubated with blocking buffer (PBS with 0.1 mg/ml BSA and 0.1% Saponin) for 10 min. Blocked cells were incubated with primary antibody (anti-Nkx2.1) in blocking buffer for 30 min. After washing with PBS, we incubated the cells with Alexa 647-conjugated secondary antibodies (1:1000) for 15 min. As a control, some samples were incubated only with secondary antibody. After washing with PBS, we suspended the cells in blocking buffer and analyzed them using the FACSAria. Flowjo software was used to analyze raw data. Ten thousand cells were recorded per analysis. As shown in Fig. 4a, more than 80% of the total cells were induced into an MGE phenotype by MGE induction conditions, as indicated by Nkx2.1 positivity (79.5 ± 1.8% Nkx2.1+ cells, n = 3). These results were in accordance with immunocytochemical characterization of cells, obtained from three different hPSC lines, using the same antibody. All PSC lines that were tested generated enriched MGE cell populations (Fig. 4b–d, Table 4).
