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. 2020 Sep 16;10:15209. doi: 10.1038/s41598-020-72214-0

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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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Differentiation and characterization of mixed 2D primary brain cultures. (A) NPCs were differentiated into either single culture or mixed cultures according to the protocol described and characterized for expression of cell specific markers by immunofluorescence. Representative images comparing single cell and mixed culture differentiation are depicted. Neurons were stained with βIII Tubulin Tuj1 (green) and astrocytes were stained with GFAP (red) and nucleus with DAPI (blue) at week 2, 4 and 6 post differentiation. Scale bar, 50 μm. (B,C) Time-course RNA analysis of the neuronal marker βIII Tubulin expression during differentiation in monocultures (B) compared to mixed culture (C) by qRT-PCR. (D,E) Time-course analysis of GFAP RNA, astrocyte marker expression during differentiation in monocultures (D) compared to mixed culture (E) by qRT-PCR. Fold change was calculated by normalizing expression level in undifferentiated NPCs in (N = 3) independent experiments.