Flow Cytometry Analysis of Maturation in Stem Cells

Cells were thawed as previously described^{21 (link)}. Cells were centrifuged and stained with GhostDye510 (Tonbo Biosciences), fixed with 4% formaldehyde, and washed with wash buffer (2% FBS in DPBS). Cells were stained with primary antibodies in 1× BD Perm/Wash (BD Biosciences) +0.2% Triton X-100 (except for Map2 stain, which did not contain Triton X-100) at 4 °C (see Supplementary Table 2 for list of antibodies and dilutions), and labeled with secondary antibodies (where applicable) at room temp. Flow cytometry was performed on a MACSQuant® Analyzer 10 flow cytometer (Miltenyi Biotec). Three biological replicates were analyzed for each maturation time point. Gating strategies are depicted in Supplementary Fig. 4.