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. 2014 Feb 7;6(2):e00137. doi: 10.1042/AN20120041

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© 2014 The author(s) has paid for this article to be freely available under the terms of the Creative Commons Attribution Licence (CC-BY)(http://creativecommons.org/licenses/by/3.0/) which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (CC-BY) (http://creativecommons.org/licenses/by/3.0/) which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.

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Spheroids were grown for 7–10 days in 2% O₂, 5% CO₂, 93% N₂ and then gently dissociated into single cells and plated on to PDL-coated chamber slides. Cells were infected with 50 CFU pNIT retrovirus for 1 day followed by growth in N2B2+0.5% FBS for 7 days after which cells were fixed in 4% PFA and triple-stained for GFP-green (A1, B1, C1), GFAP-blue (A2, B2, C2) and O4-red (A3, B3, C3). Clones were classified as astrocyte - only clones (A4); oligodendrocyte - only clones (B4); or mixed clones (C4). Scale bar represents 40 μm (A and B) and 20 μm (C). (D) Quantification of two independent experiments in which clonal analyses were performed after infection with 1×10⁵ CFU of the pNIT reporter replication-deficient retrovirus in N2B2 differentiation media (white) or in ProN/B104 CM (black).