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Adult Mouse Cardiac Myocyte Isolation - Procedure

Procedure

I. Langendorf Perfusion System

1. Turn on the water bath and water heater.
2. Warm 75 ml of the enzyme solution and 75 ml of Ca²⁺ free Tyrode to 36.9 C in the water bath under the heart chamber.
3. Prime the perfusion system with the Ca²⁺ free Tyrode solution.
4. Turn on the O₂ supply and bubble the Ca²⁺ free Tyrode solution.
5. The temperature in the heart chamber should be 36.4 36.9 C.

II. Mouse Preparation

1. Inject the mouse with 0.05 ml of heparin IP (1000 U/ml).
2. Wait 10 minutes to allow the heparin to be absorbed and circulate and then anesthetize the mouse with 0.6 ml of avertin IP.

III. Heart Perfusion and Myocyte Isolation

1. Check the mouse for lack of response to limb and tail pinch and then quickly excise the mouse heart.
2. Place the heart in a 30 ml beaker of Ca²⁺ Tyrode solution. Shake and massage the heart a few times in this solution to remove the blood.
3. Tranfer the heart to a dish of Ca²⁺ Tyrode solution and remove all the fat and connective tissue.
4. Cannulate the heart by the aorta.
5. Perfuse the heart for 2 minutes with Ca²⁺ free Tyrode solution.
6. Then perfuse the heart for 8 minutes with the enzyme solution.
7. Make sure the temperature of the perfusate is between 36.0 to 36.9 C.
8. After the digestion, the heart should be palpably flaccid. Turn off the oxygen and heater. Cut the heart from the cannula.

IV. Myocyte Isolation

1. Briefly place the heart into a 30 ml beaker of Ca²⁺ free Tyrode solution.
2. Transfer the heart to a small glass dish of Ca²⁺ free Tyrode solution with albumin. This solution should be portioned from the 75 ml mixed up at start of the procedure.
3. In this dish cut away and remove the chambers that are not to be used.
4. Cut the remaining chambers into chunks [a few mm on each edge] and then gently titurate them through a large tip transfer pipette.
5. Repeat the tituration two more times using a smaller tipped pipette each time [a blue 1 ml tip followed by a yellow 0.2 ml tip].
6. For atrial tissue strain the pieces over a 50 ml tube of Ca²⁺ Tyrode and store them at 4 C until use for patch clamp recordings.
7. For ventricular tissue strain the pieces over a 50 ml tube of Ca²⁺ free Tyrode solution with albumin. Let the cells settle for 15 minutes in the tube.
8. Slowly transfer the cells to the calcium containing buffers. Let the settle for at least 10 minutes in each of the tubes. The cells will remain in the 1.2 mM Ca²⁺ Tyrode solution at 4 C until used for patch clamp recordings.
9. Clean the perfusion with cold deionized water and then boiling water after each isolation.

 

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