Fig. 4

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.