Objective: To investigate the factors affecting the viability and Ca 2+ tolerance of isolated rats' cardiac myocytes for patch clamp research. Methods: Hearts were firstly perfused by the Langendorff perfusion apparatus with normal Tyrode's solution, then with Ca 2+-free Tyrode's solution and subsequently with low Ca 2+ enzyme solution containing collagenase 0.1-0.2 g/L. All the solutions were saturated with oxygen and the perfusion temperature was kept at 37 ℃. Finally hearts were washed by Ca 2+-free Tyrode's solution, after which the ventricles were minced into small pieces in KB solution, dispersed and filtered. The isolated myocytes were stored in KB solution at room temperature for 1 h and recovered to normal calcium concentration before patch clamp experiments.Results: When all the factors such as water, enzyme, Ca 2+,pH, and oxygen were well controlled, the well constructed and rod-like cardiac myocytes with a yielding rate of 30%-50% came out.Conclusion: All the factors should be well controlled, which ensured the isolated cells Ca 2+ tolerant and appropriate for patch clamp experiments.
