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. 2019 Apr 12;9:5977. doi: 10.1038/s41598-019-42439-9

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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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Quantification of neural and vascular markers by flow cytometry and histology images of late stage hybrid spheroids. Flow cytometry histograms for day 21 iEC spheroids, iNPC spheroids, or tri-cultured spheroids for (A) β-tubulin III; and (B) CD31. Black line: negative control; red line: marker of interest. The percentage indicates the positive cells versus total cells. (C) Flow cytometry quantification of β-tubulin III (β-tub III, n = 3) and (D) CD31 (n = 3). *Indicates p < 0.05 for the different test conditions. (E) Images of histological sections of late stage spheroids (total day 47) showed the expression of cortical layer markers: TBR1 (red)/SATB2 (green), BRN2 (red)/SATB2 (green). SATB2: cortical superficial layer II-IV. Hoechst: blue. The dashed lines indicate the locations of spheroids and the cortical layers. Scale bar: 100 μm. (F) Confocal images of histological sections of spheroids (total day 47) for β-tubulin III (green) and vascular marker CD31 (red). Scale bar: 50 μm.