For analysis of percentages of CMs in the heart, whole E17.5 ventricles were dissociated from control and Prox1ΔLEC/ΔLEC hearts using the Pierce Primary Cardiomyocyte Isolation Kit (Thermo Fisher). Cells were fixed and permeabilized using a Permeabilization kit for intracellular staining (eBioscience) following the manufacturer’s instruction. Cells were then incubated with Cy3-conjugated mouse anti-cardiac Troponin C antibody (Abcam, ab45931, 1:100) and Hoechst 33342 (Invitrogen, 1:1000) at room temperature for 1 h. Cells were washed and percentage of cTnC+ CMs was determined after 20,000 total cell counts by flow cytometry. Percentage of polyploidy CMs was determined by Hoechst 33342 intensity. Flow data was collected using the flow software BD FACS Diva 8.0.3 and analyzed by FlowJo v10.
For analysis of the purity of differentiated hiPSC-CM, dissociated CMs were fixed with 4% PFA and permeabilized using 0.5% saponin. Cells were then incubated with 647-conjugated mouse anti-cardiac TNNT2 antibody (BD Biosciences, clone 13-11, 1:200) for 1 h. Cells were washed and percentage of TNNT2+ CM was determined after 10,000 total cell counts by flow cytometry.
For analysis of the purity of differentiated hiPSC-CM, dissociated CMs were fixed with 4% PFA and permeabilized using 0.5% saponin. Cells were then incubated with 647-conjugated mouse anti-cardiac TNNT2 antibody (BD Biosciences, clone 13-11, 1:200) for 1 h. Cells were washed and percentage of TNNT2+ CM was determined after 10,000 total cell counts by flow cytometry.