An innovative sequence of hypoxia-reoxygenation on adult mouse cardiomyocytes in suspension to perform multilabeling analysis by flow cytometry

Introduction Because cardiovascular diseases still represent the leading cause of death worldwide, a better understanding of the underlying physiopathological mechanisms is therefore needed. In vitro cellular models participate to decipher the molecular mechanisms, especially adult mouse cardiomyocytes. Unfortunately, due to their high fragilitity as well as their size (150 μm), flow cytometry is usually not performed and mainly conventional techniques are used like cell imaging, which is time and animal-consuming and causes low statistical power. Methods and results Here, we described a new, simple and rapid one-day protocol in living adult mouse cardiomyocytes submitted to in suspension hypoxia-reoxygenation to allow multilabeling analysis by flow cytometry. Our method enables the measurement of several physiological parameters thanks to fluorescent probes labeling, assessing notably cell viability (Propidium Iodide, Calcein-AM and Sytox Green), mitochondrial membrane potential [DilC1(5), TMRM], reactive oxygen species (ROS) production (MitoSOX Red) and mitochondrial mass (MitoTracker Deep Red). Additionally, our model can be used to perform pharmacological screening illustrated here by the protective treatment via cyclosporine A (CsA) as a test to assess robustness and sensitivity of the utilized methods. Conclusion In summary, our new hypoxia-reoxygenation sequence in suspension will offer a high-speed quantitative multilabeling analysis of adult mouse cardiomyocytes, which can be extended to various cellular stress challenges (oxidative, inflammation) or pharmacological screening.