I have recently started to do outside-out patch clamping with an TET-ON inducible HEK-293 cell line. I was curious if people had any tips for this? I have no issues getting a seal and a break-in, but when I try to pull the pipette away from the cell I usually lose the seal (if the cell doesn't come up with the pipette).
More specifically; any suggestions on pipette resistance, how to pull the pipette from the cell (slow or fast), what should I be looking for on the amplifier's membrane test (lower capacitance, but with a good seal), etc? Thanks in advance!
1/ pip: Res.: The smaller, the higher chances to get a single channel recording
2/ I pull as slow as possible
3/ as you pull, your capa will decrease to a minimum. You do correct for Capa before going WC? if you do, then you will get back to very similar values when you pull the patch.
4/ You membrane test should indicate at least > 2 GOhms or higher. The higher value, the lower signal noise you will have.
Hello Joshua Kaplan. May I know how your outside-out recording went? I have recently started doing outside-out recording but I couldn't make the outside-out patch after making whole-cell. When I try to pull the pipette, after few moments the leak increases, and I lose the cell. In general, after making the whole cell I try to lift up the pipet a little and retract a little, lift up and retract.... and monitor the decrease in capacitive transient.
Would you please share any tips from your experience on how I can get the outside-out patch?
btw I am recording sodium current and getting a perfect giga seal is not always possible. So in my whole cell situation, some leak current is always present.
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