Flow cytometric analyses for ENSCs, gliocytes and neurons [25]

Flow cytometry was used to define the neural cell populations generated. Colonies in culture wells were first dissociated by incubating in pre-warmed (37 °C) in 0.025% trypsin for 4 min and then quenched by adding twice the volume of flow buffer (2% fetal bovine serum in PBS). Cell suspension was gently triturated by a microliter pipet to prepare a single cell suspension. Following PBS wash and centrifugation (at 220×g for 5 min at 25 °C), cell pellets were resuspended in an appropriate volume for staining.

For intracellular staining, cells were fixed, permeabilized and washed with BD Cytofix/Cytoperm™ Kit (BD Biosciences). Samples were first stained with anti-Tubulin β3 (TUBB3 or TUj1, expressed in neurons) and anti-p75 neurotrophin receptor (p75, expressed in ENSCs) primary antibodies, followed by appropriated secondary antibodies conjugated with fluorescence. Following vigorous wash with washing buffer, cell were finally treated with fluorescence-conjugated primary antibodies against S100 calcium-binding protein B (S100b expressed in gliocytes, C-3, Santa Cruz Biotechnology). After the last wash, cells were acquired by BD FACSCantoTM II and analyzed with BD FACSDiva software.