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. 2019 May 2;9:6811. doi: 10.1038/s41598-019-43080-2

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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mOrange+ DA neurons are amenable to FACS-mediated purification and survive after sorting. (A) Experimental procedure followed for FACS-sorting and subsequent re-plating of mOrange+ DAn. (B) Cytograms from FACS of mOrange+ cells from differentiated TH-mOrange iPSC. Purified cells were seeded after sorting and re-analyzed after 7 days. Cytograms are representative of 2 independent experiments. (C,D) Immunofluorescence analysis of mOrange+ DAn sorted and reseeded on Matrigel for 1 (C) and 7 (D) days. One day post-sorting, living neurons were first imaged and then fixed and stained with antibodies against TUJ1 (green), TH (grey) and mOrange (red). The same combination of antibodies was used to stain neurons cultured for 7 days after sorting. Nuclei were counterstained with DAPI. Scale bars, 20 µm and 10 µm for the large image and the inset, respectively. (E) Quantitative analysis of the percentage of cells staining positive for TH 7 days after sorting. Data is the average ± S.E.M. of two or three independent experiments. Sorted mOrange+ cells, 1232 cells from 2 independent experiments; sorted mOrange- cells, 1020 cells from 3 independent experiments; unsorted cells, 679 cells from 2 independent experiments. Asterisk denotes statistically significant differences (*p < 0.05).