Hi,
I would like to measure whole-cell currents in HEK cells. I have no problem achieving a GOhm seal, and sometimes I also succeed in going whole cell. But, mostly, when I try to rupture the membrane, the seal breaks. I used both suction and the ZAP function of my Axopatch 200B amplifier, to no avail. My pipette resistances are btw. 4 and 5 MOhms, as these are supposed to be optimal for whole-cell recording.
I would be very grateful for any suggestions how to solve this problem.
Hi
Ensure that the pipette tip is very smooth and has a fine, uniform tip, ideally between 1-2 µm in diameter. Rough or uneven tips can increase the likelihood of seal instability, especially when trying to rupture the membrane.
Double-check that your internal solution is well-prepared and that the resistance is stable. On your Axopatch 200B, you can adjust the ZAP function settings, including the frequency and intensity. If you’re experiencing seal breakage, it might be helpful to try a lower-intensity ZAP pulse to avoid too much pressure on the membrane.