Whole Cells in Patch Clamp

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núria
núria's picture
Whole Cells in Patch Clamp

We have a patch clamp setup with a HEKA EPC 10 USB amplifier and we have a very frustrating problem: we achieve good Gseals with transfected cells but when we want to open to a whole cell, the seal is always lost and the pulse goes down.
 
We use HEK cells transiently transfected with a K+ channel. It seems that when cells are “driving” a current the seal is always broken before to achieve good a whole-cell. But it works well with HEK cells non-transfected, HEK cells transiently-transfected with a regulatory subunit KCNE1 (that is not an ion channel) and HEK cells with YFP. But not when there is a channel on the membrane. Do you think it makes any sense?!?
 
We are so desperate that we tested everything we could moved (our transfected cells, solutions, polished pipettes, holder, headstage, probe cable, amplifier, as well as both electrodes) to a setup from a colleague and we did a lot of recordings! So, all these parts are not the problem.
 
We even moved the whole setup to another part of our building to see if there is some kind of interference associated to our patch clamp room... and we still have the same problem to achieve whole-cells.
 
The only parts of our setup we haven't changed are the micromanipulator and the tubing (but we have the same holder and therefore, the same diameter that our friends). We tred it by mouth and by a 1 mL syringe (0.1 mL by 0.1 mL) although we always did the whole cells by mouth before, and it worked like this also in our colleague's set up...  
 
Do you have any other idea? Thanks so much for your help!
 
núria
 
The noise is around 3 pA once the pipette touches the cell surface. We should improve it but we don’t think it is a big deal if we do whole-cell recordings. In fact, out transfected cells showed around 10-20 nA of amplitude, and it worked before!!!