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. 2018 Aug 13;8:12028. doi: 10.1038/s41598-018-30537-z

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© The Author(s) 2018

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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Cytometric and immunofluorescent analyses of the expression and localization of the early cardiac markers GATA4 and Nkx2.5 during the different phases of the differentiation protocol. ^{Cardiopoietic}AF: before the differentiation process; mesodermal induction: after 24 hrs of activin A and BMP4; myocardial induction: after 72 hrs of VEGF; cardiomyocytes: after 15 days of differentiation. (a) Flow cytometry histograms: gray line, isotype control; blue line, ^{Cardiopoietic}AF; green line, mesodermal induction; yellow line, myocardial induction; red line, cardiomyocytes obtained after 15 days of differentiation. (b) % of positive cells and mean fluorescence intensity at each differentiation step. Data are expressed as the mean ± SD. (c) Immunofluorescent staining for GATA4 (green), Nkx2.5 (red) and nuclei (blue) during the different phases of the differentiation protocol. The bottom line shows the merged fluorescence, as indicated. Scale bar: 10 μm. Data are representative of seven independent experiments.