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. 2020 Apr 14;10:6272. doi: 10.1038/s41598-020-62925-9

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© The Author(s) 2020

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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A panel of representative immunostaining images of 16 day old mature neurons derived from (upper) 2D monolayer and (lower) 3D neurosphere cultures. Monolayer-derived neurons labelled with (A,A′) DAPI, confirming their maturation by expressing (B,B′) Tuj1 and further confirming their cortical linage being positive for (D,D′) Tbr1 and with modest expression for (C,C′) Ctip2. (E,E′) Merged images. Neurosphere-derived neurons labelled with (F,F′) DAPI, showing their positivity for (G,G′) Tuj1 and (H,H′) Ctip2 but not (I,I′) Tbr1. (J,J′) Merged images. Flow cytometry comparative analysis of (K–N) Tbr1 and (O–R) Ctip2 expressions between monolayer- and neurosphere-derived neurons with their corresponding undifferentiated counterparts. A comparative analysis of the positively stained monolayer- and neurosphere-derived neurons for (S) Tbr1 and (T) Ctip2 comparing to their undifferentiated counterparts. Log-2-fold change in the transcriptional expression of (U) Sox5 and (V) Fezf2 supporting the deep layer cortical identity. Data represents mean ± SEM, n = 5, Students t-test *p < 0.05, ***p < 0.001. Scale bars = 100 um.