Extended Data Figure 2. Identification of non-myocytes from the hearts of Kit^+/Cre × R-GFP mice.

Kit^+/Cre × R-GFP mice were harvested at 6 weeks of age (constitutive lineage labeling the entire time), although MI was performed at week 4 to induce greater vascular remodeling and potentially more c-kit lineage recruitment over the next 2 weeks. a, Hearts were then collected at week 6 and subjected to immunohistochemistry with a pool of antibodies for CD31, CD34, CD45 and CD3 in red, while the green channel was for eGFP expression from the recombined R-GFP reporter allele due to Kit-Cre lineage expression. The white arrowheads show endothelial cells that are not contiguous with the underlying network, although most of the endothelial cells are from the c-kit lineage when the red and green channels are compared. The white arrow shows a cardiomyocyte that lacks red staining, while the yellow arrows show 2 areas with relatively large cells that are eGFP⁺ and could be mistaken for a cardiomyocyte, although they are also positive for the non-myocyte marker panel of antibodies. b, c, Spread of cells isolated from hearts of 8 week-old Kit^+/Cre × R-GFP mice at baseline that were subjected to immunocytochemistry for the indicated markers. The large white arrow in panel b shows an eGFP⁺ (green) cardiomyocyte that also co-stains with sarcomeric α-actin (red). The smaller arrows show eGFP⁺ non-myocytes, which in panel c, were subject to staining with a cocktail of antibodies again for CD31, CD34, CD45 and CD3 (all in red). This analysis identifies nearly all of the non-myocytes in these cell spreads. The very last image in panel c shows a fourth channel with higher gain so that the underlying cardiomyocytes (CMs) autofluoresce (in white) to show the mixed nature of the spread cells. Nuclei were stained blue with DAPI in the indicated panels.