iPS cells were maintained and differentiated as previously described (see Supplementary data ) 37 (link). At day 18 neural precursor cells (NPCs) were isolated using Anti-PSA-NCAM MicroBeads (30-092-966¸ Miltenyi Biotec) according to the manufacturer’s protocol. Sorted cells were fluorescently stained with an Anti-IgM antibody conjugated with PE dye (130-095-908; Miltenyi Biotec) and analysed by flow cytometry using a GUAVA EasyCyte 6-2L instrument to assess purity of the recovered population. Unlabelled cells were used as negative control. Cultures were harvested at day 70 for analysis of mature neurons. To obtain a more pure population of neurons for qRT-PCR, mature cultures were sorted using a CD24 MicroBead Kit (130-095-951, Miltenyi Biotec)39 (link). Sorted neurons were plated on poly-ornithine and laminin coated slides and allowed to attach for 48 hours before being stained for β-III-Tubulin (Chemicon) to confirm their identity.
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Efficient Isolation and Characterization of Mature Neurons
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Other organizations : University of Siena, University of California, Davis, University of Turin
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Variable analysis
independent variables
- Differentiation of iPS cells to obtain neural precursor cells (NPCs)
- Purity of recovered NPC population assessed by flow cytometry
- Maturation of neurons in culture at day 70
- Expression of neuronal markers in sorted mature neurons
- Unlabelled cells used as negative control for flow cytometry
- Positive control: Not explicitly mentioned.
- Negative control: Unlabelled cells used for flow cytometry analysis.
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