Pe conjugated mouse anti cd24

The human ES (H9G1) and HUES3 ISL1-RFP reporter cell lines were differentiated to embryoid bodies as described by Bu et al.4 (link) Briefly, human ESCs were treated with collagenase (1 mg ml⁻¹; Gibco), and then suspended in differentiation medium containing BMP4 (20 ng ml⁻¹), activin A (10 ng ml⁻¹) and WNT3A (100 ng ml⁻¹; all from R&D Systems) before transferring to six-well ultra low cluster plates (Costar), and cultured at 37 °C. ISL1-expressing cells (GFP- or RFP-positive) were typically observed on day 7 after differentiation. The embryoid bodies were dissociated with accutase (Life Technologies). Single-cell suspension was obtained, and the cells were stained with PE-conjugated mouse anti-CD24 (BD Bioscience) and mouse anti-human Pan-Neuronal marker (Millipore) at concentration of 1:100 dilution, followed by donkey anti-mouse IgG Alexa 647 at 1:1,000 dilution (Invitrogen). Isolation of ISL⁺ CVP was achieved by fluorescence-activated cell sorting for GFP⁺/CD24⁻/Pan-Neuronal⁻ or RFP⁺/CD24⁻/Pan-Neuronal⁻ cells. Single-sorted cells were seeded onto irradiated feeder layer (MEF) and cultured with CGM medium supplemented with EDN1 and WNT3A.