Human neural lineage analysis kit

For FACS analysis, cells were harvested and washed. Cells were fixed and permeabilized with a human neural lineage analysis kit (BD Biosciences) according to the manufacturer’s instruction. 1 × 10⁵ cells were stained with Alexa 488 (for MAP2 and Tuj1) or 647-conjugated (for Nestin and GFAP) antibodies against MAP2, Tuj1, Nestin, and GFAP (BD Biosciences) on ice for 30 minutes. The fluorescence intensity was measured on a FACSCalibur™ (BD, San Joes, CA, USA), and the data were analyzed using FLOWJO software (Tree Star, Inc., Ashland, OR, USA).

For flow cytometric analysis of EB-NPCs, cells were collected using Accutase and stained with mouse anti-Nestin, mouse anti-human Sox1, and mouse anti-Sox2 per manufacturer’s instructions using the Human Neural Lineage Analysis kit (BD). For immunofluorescence microscopy, EB-NPCs were seeded on Geltrex-coated slides, fixed with 4% paraformaldehyde, and stained with rat anti-Nestin (1:500; Millipore), rabbit anti-Sox2 (1:200; Epitomics), or rabbit anti-Pax 6 (1:50; BioLegend) before addition of respective secondary antibodies (goat anti-rabbit AlexaFluor 568 and goat anti-rat AlexaFluor 488; both from Thermo Fisher). For analysis of multipotency, EB-NPCs were cultured in neuronal differentiation medium (Neurobasal, 1X B27, 1X GlutaMAX), astrocyte differentiation medium (DMEM [Thermo Fisher], 1X N2, 1X GlutaMAX, 1% FBS [Atlanta Biologicals]), or oligodendrocyte differentiation medium (Neurobasal, 1X B27, GlutaMAX, 30 ng/ml T3 [Sigma]) for at least 14 days before being fixed with 4% paraformaldehyde and stained with mouse anti-beta-III-tubulin (1:500; Abcam), rabbit anti-GFAP (1:200; Thermo Fisher), rabbit anti-NG2 (1:200; Chemicon), or rabbit anti-Olig2 (1:100; Abcam). Slides were cover-slipped and mounted using VectaShield Hard Set Mounting Medium with DAPI (Vector Labs), and all images were captured using a Nikon Eclipse Ti inverted microscope.