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Figure 3 (see Plate 5) FACS®- and Immunopannlng-based lineage selection of mES cell-derived neurons. (A, B) FACSorting of Tal-EGFP-positive cells results In efficient enrichment of neuronal cells, yielding purities of >98% p-III-tubulin-positive cells. (A) EGFP expression (green) and p-III-tubulin immunofluorescence (red) in the sorted cell population 1d after plating. The inset in (A) shows a high-power view. (B) Corresponding nuclear Hoechst stains. (C, D) Following a 2d period of growth factor withdrawal, immunopanning of ESNPs with an antibody to PSA-NCAM yields purities of >95% p-III-tubulin-positive neurons.

(C) PSA-NCAM-positive cells (red) double labelled with an antibody to p-III-tubulin (green) 2d after immunopanning (Inset: detail). Nuclei are counterstained with Hoechst (blue).

(D) Corresponding phase-contrast micrograph. Scale bars = 50mm in C and F. Adapted from Schmandt et al. (28).

EGFP-positive neurons generated from ESNPs. Transgenes that have been used successfully to this end include Ta1-EGFP ((28); and see Figure 3A, B) and Tau-EGFP (27). The time point of FACS® has to be adapted to the kinetics of the individual reporter gene activity. Ideally, FACS® of Tal-EGFP- and Tau-EGFP-positive neurons is performed 2 d and 7 d after initiation of differentiation, respectively. Both methodologies typically yield neuronal populations with purities exceeding 90% (27, 28).

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