The protocol used for myocyte isolation is described in detail elsewhere18 (link). Briefly, animals were anesthetized with pentobarbital, and hearts rapidly removed via thoracotomy. The aorta was cannulated on a Langendorff apparatus fitted with a heating jacket circulating water at 37°C, and retrograde-perfused for 5 minutes at 8 ml/min with Tyrode’s solution. The perfusate was then switched to Tyrode’s solution containing collagen type 2 (Worthington) and protease type 14 (Sigma-Aldrich) for 7 minutes. The solution was switched to a modified Kraft-Bruhe (KB) buffer for 5 minutes. The left ventricle was minced and filtered (200μm) to yield single cells. Myocytes rested in KB buffer19 (link) for an hour before being placed in supplemented M199 ACCIT medium20 (link).
Protocol detail
Isolation of Cardiac Myocytes
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Animals
Aorta
Buffer
Cells
Hearts
Isolation
Left ventricle
Myocytes
Pentobarbital
Protease
Thoracotomy
Type 2 collagen
Corresponding Organization : Johns Hopkins Medicine
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Variable analysis
independent variables
- Perfusate switch to Tyrode's solution containing collagen type 2 and protease type 14
- Perfusate switch to a modified Kraft-Bruhe (KB) buffer
- Myocyte isolation and yield of single cells
- Anesthesia of animals with pentobarbital
- Retrograde perfusion of the aorta at 8 ml/min with Tyrode's solution for 5 minutes
- Temperature of the perfusate at 37°C
- Mincing and filtering of the left ventricle (200μm)
- Resting of myocytes in KB buffer for an hour before being placed in supplemented M199 ACCIT medium
- None specified
- None specified
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