Larsen, M. S.; Thomsen, M. B.; Zawadzki, T.

This protocol describes a Langendorff-based method for isolating intact adult mouse ventricular myocytes using syringe pump-driven perfusion. The approach retains the key physiological advantage of the conventional Langendorff technique, continuous retrograde coronary perfusion, while simplifying the overall setup. By combining retrograde aortic perfusion with widely available laboratory equipment, the method provides an accessible alternative to traditional Langendorff systems. A precision syringe pump connected to an in-line heater is used to deliver temperature- controlled, constant-flow perfusion during enzymatic digestion. In contrast to gravity-driven constant-pressure systems, constant-flow perfusion maintains stable enzyme delivery despite changes in coronary resistance that occur during tissue digestion. Use of an inline heater allows precise, rapid temperature-controlled delivery, avoiding the complexity, leak risk, thermal lag, and contamination susceptibility associated with traditional water-jacketed systems. Our setup reduces variability in perfusion rate and minimizes susceptibility to occlusion, flow interruption, or compliance-related artifacts, enhancing reproducibility. The method consistently yields adult ventricular myocytes with high viability (>70% rod-shaped, calcium-tolerant), enabling a broad range of functional analyses including electrophysiology, contractile performance and calcium handling. Step-by-step instructions, troubleshooting guidance, and anticipated outcomes are provided to facilitate adoption in laboratories without dedicated isolated-heart perfusion infrastructure.