Protocol for simultaneous isolation of high-quality & high-quantity cardiomyocytes and non-myocytes

Here, we present a modified isolation protocol utilizing Langendorff perfusion and subsequent enzymatic digestion to isolate cardiomyocytes and non-myocytes cells including immune cells with high quality and high quantity. In addition, we present a detailed protocol using fluorescence-activated cell sorting (FACS) for identification of live non-myocyte cell types. Advantages from our isolation approach include I) Isolation of immune cells together with all other cardiac cells from the same heart, which allows for investigations of multiple cell types simultaneously. This approach will also reduce the animal number to be used if two or more cell types are analyzed; II) Establishment of a standardized protocol with consistent enzyme composition and concentration, which will allow the comparison of data from different laboratories; III) Cardiomyocytes and non-myocyte cells are purified using bovine serum albumin (BSA) solutions that are easy to prepare and have minimal effects on cell viability (compared to Percoll gradient); and iv) Isolated cells can be used for further experiments such as single cell RNA sequencing, gene expression profiling, functional and biochemical assays, or can be cultured for various in vitro experiments.

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