Assays to quantify myocardial renewal rely on the accurate identification of cardiomyocyte nuclei. We previously ¹⁴C birth dated human cardiomyocytes based on the nuclear localization of cTroponins T and I. A recent report by Kajstura et al. suggested that cTroponin I is only localized to the nucleus in a senescent subpopulation of cardiomyocytes, implying that ¹⁴C birth dating of cTroponin T and I positive cell populations underestimates cardiomyocyte renewal in humans. We show here that the isolation of cell nuclei from the heart by flow cytometry with antibodies against cardiac Troponins T and I, as well as pericentriolar material 1 (PCM-1), allows for isolation of close to all cardiomyocyte nuclei, based on ploidy and marker expression. We also present a reassessment of cardiomyocyte ploidy, which has important implications for the analysis of cell turnover, and iododeoxyuridine (IdU) incorporation data. These data provide the foundation for reliable analysis of cardiomyocyte turnover in humans.