Considering that you have success with patching, instead of breaking the membrane, an option is to perforate it with the use of amphotericin. This has the advantage of maintaining the intracellular ionic environment almost intact.

Thanks, Jorge Hidalgo

As you recommend. it is an alternative way.

Is this problem only related with patchling or does the cell isolation matter, too?

Does anyone give suggestions?

Hi Masaya, I bet you know this very well, but the outcome of the cell isolation does influence patch clamp efficiency. That is, when the cells are underdigested, it becomes difficult or impossible to break through. Unfortunately it is difficult to give any specific advice on the isolation procedure, as there is a bunch of conditions that can not be standardized, such as the tissue itself, or the collagenase activity which often changes from batch to batch etc. When you end up with underdigested cells, you can still play around with the shape and quality of the patch pipettes. If you fire polish regularly, you might want to consider using unpolished ones. Also, try to use as large tip diameters as possible without compromising seal formation. The larger the tip diameter, the easier break through becomes. Good luck!