We are unable to patch adult ventricular cardiomyocytes from mice. Our initial seal is terrible and leaky. Ca++ floods in and the cell begins to beat. We get ~ 1 million nice rod-shaped, striated cardiomyocytes from our isolation. Anyone have any experience patch clamping these cells? Any advice on getting a good seal would be appreciated.
Mouse ventricular myocytes have to be handle very carefully since they can overload with calcium very easily. I would recommand also to use 3-5 mOhm pipettes, to applie a bit of positive pressure to the pipette before sealing. Then when seal is achieved wait before going whole cell.
It is true that having a good yield of good looking cells is does not mean they are OK for patch-clamp. Try maybe to use a differnt protocole for isolation of the cells. I seed cells on coverslips coated with laminin, wait for 30-45 min (in icubator) and then add more solution (by that time the cells should be adherent to the coverslip). Try not to add too many cells to the coverslips. Avoid to patch cells that are already beating.
..And anyone doing perforeted patch in adult mouse ventricular myocytes?
We have also difficulties to get a good long seal. For perforeted-patch (amphotericin) we use 1-2 MOhm pipette resistance and we do not polish them because we lack a polisher. Is fire polish a critical step?
Thanks a lot for your advice
- Badges
- Science method