| Summary: | Summary: Several protocols now support efficient differentiation of human pluripotent stem cells to cardiomyocytes (hPSC-CMs) but these still indicate line-to-line variability. As the number of studies implementing this technology expands, accurate assessment of cell identity is paramount to well-defined studies that can be replicated among laboratories. While flow cytometry is apt for routine assessment, a standardized protocol for assessing cardiomyocyte identity has not yet been established. Therefore, the current study leveraged targeted mass spectrometry to confirm the presence of troponin proteins in day 25 hPSC-CMs and systematically evaluated multiple anti-troponin antibodies and sample preparation protocols for their suitability in assessing cardiomyocyte identity. Results demonstrate challenges to interpreting data generated by published methods and inform the development of a robust protocol for routine assessment of hPSC-CMs. The data, workflow for antibody evaluation, and standardized protocol described here should benefit investigators new to this field and those with expertise in hPSC-CM differentiation. : Waas and colleagues demonstrate pitfalls with popular antibodies and sample preparation conditions commonly used for the assessment of cardiomyocyte identity within differentiation cultures. By using a rigorous fit-for-purpose workflow, the authors developed and validated a comprehensive protocol to accurately assess cardiomyocyte identity within hPSC-CM cultures. The new protocol includes stepwise instructions to facilitate its implementation by experts and novices alike. Keywords: quality control, flow cytometry, troponin, cardiomyocytes, mass spectrometry, standard operating protocol
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