Embryonic epicardial cells and cardiomyocytes were isolated using a previously published protocol (Burns and MacRae, 2006). We extracted hearts from approximately 500 transgenic embryos harboring tcf21:DsRed2; gata5:EGFP (Kikuchi et al., 2011) or cmlc2:DsRed2 (Kikuchi et al., 2010). In tcf21:DsRed2; gata5:EGFP embryos, EGFP and DsRed2 colocalization was restricted to the epicardium (Kikuchi et al., 2011), and this effect enabled us to isolate highly purified epicardial cells (Figure 4A). The extracted hearts were placed in a Petri dish containing ice-cold 1× Hanks’ Balanced Salt Solution (HBSS) and examined using an MVX10 microscope. To prepare single-cell suspensions, hearts expressing the fluorescent reporter were collected with a pipette and transferred to a 1.5-ml tube containing 1× HBSS with 1 mg/ml collagenase type 2 (Worthington Biochemical, Lakewood, NJ). The samples were incubated for 40 min at room temperature and mixed by gentle pipetting every 10 min. The dissociated cells were washed and resuspended in ice-cold 1× HBSS containing 5% fetal bovine serum (FBS).