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. 2017 Jun 2;7:2687. doi: 10.1038/s41598-017-02461-1

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© The Author(s) 2017

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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Culture of ventricular endothelial cells isolated from adult Tg(kdrl:EGFP) zebrafish. (a–b) Brightfield and fluorescence images of cultured cells (60 h after seeding) on a fibronectin coated culture dish without a preplating step (a) or after a 4 h preplating step (b). Black arrowheads, non-endothelial cells; BF- Brightfield. (c) Example of brightfield and fluorescence images (green for EGFP positive endothelial cells, and blue for nuclei (Hoechst 33342)) of isolated live ventricular cells on the preplate, after 4 h preplating. (d) Example of cultured cardiac endothelial cells after 1^st passage, stained for EGFP (green), rhodamine phalloidin (all cell types, red), and DAPI (nuclei, blue). (e) Quantification of endothelial cells to total cells after 1^st passage, showing high purity of the cultures. (n = 3, mean ± SEM). (f) RT-qPCR analysis using cardiomyocyte (myl7), fibroblast (vim), smooth muscle cell (acta2), epicardial (wt1b), and endothelial (kdr) markers (n = 3, mean ± SEM). Marker gene mRNA expression levels relative to α-tubulin were calculated using the ΔCt method (ct values are indicated in the Supplemental Table 2). Expression is relative to the individual marker’s expression in whole cardiac ventricles. All non-endothelial markers, besides wt1b, are depleted in the isolated cell population. Concomitantly, the isolated cells show enrichment for the endothelial marker kdr. One way ANOVA followed by Bonferroni’s post-hoc test (GraphPad Prism) was performed to evaluate statistical significance of differences. P < 0.05 was considered statistically significant. *** corresponds to P < 0.001.