Multiparametric Stem Cell Characterization

hiPSCs were labeled with TRA-1-60-488 (5 μl per 1×10⁶ cells, BioLegend #330613) and SSEA4-647 (5 μl per 1×10⁶ cells, BioLegend #33407) in 1% (w/v) BSA for 45 min at 4 °C before being washed with 1× PBS and resuspended in FACS buffer (1× PBS (no Mg²⁺/Ca²) containing 1% (v/v) BSA and TO-PRO®3 (1 μM, ThermoFisher Scientific) and filtered using a 40 μm filter (BD Biosciences). NPCs were dissociated using Accutase, fixed for 10 min in 4% paraformaldehyde (PFA), permeabilized and blocked with 0.5% (v/v) Triton (Sigma)/1% (w/v) bovine serum albumin (BSA, Sigma) in PBS and labeled with NESTIN-647 (20 μl per 1×10⁶ cells, BD Biosciences #560393) and SOX2-488 (0.25 μg per 1×10⁶ cells, BioLegend #656110) antibodies overnight at 4 °C before being washed with PBS and resuspended in FACS buffer (1× PBS (no Mg²⁺/Ca²) containing 1% (v/v) BSA and TO-PRO®3 (1 μM, ThermoFisher Scientific) and filtered using a 40 μm filter (BD Biosciences). Cytometry was performed using a LSR-II or FACS Canto (BD Biosciences) and analysis was performed using Flowjo (v8.7.3, Treestar).