Difficulty in patch clamping neurons

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VG30's picture
Difficulty in patch clamping neurons

 I have been trying to patch clamp adult dorsal root ganglion neurons for a while now but can't form a gigaseal. My pipette resistance varies between 2-4 Mohms and I very lightly touch the cells before applying suction. The resistance increases upto 20-100 Mohms but I don't get a gigaohm seal. Would really appreciate if somebody could give me tips on how to patch clamp these cells successfully. My extracellular and intracellular solutions are fairly standard (i.e. high Na outside and high K inside). This is the first time I am trying to patch clamp neurons. Could I be doing something wrong?
Thanks in advance.

mdmbron's picture
Are you applying positive

Are you applying positive pressure to the electrode tip before contacting the cell? I usually have between 20-30mbar of positive presure coming out of the electrode tip. I lower the electrode until I can see a dimple in the cell membrane at which point I release the positive pressure which is often enough to form the gigaseal instantly. If the gigaseal does not form instantly I sometimes apply brief positive pressure followed by prolonged, but gentle, negative pressure to the electrode. 

How old are the adult DRG you are using? Olders membranes are more rigid and it may take more positive pressure and/or greater electrode force to achieve the dimple. In this case, after forming the gigaseal, I lift the electrode 1-2 microns to relieve the pressure on the cell. 

Also what are the Mg+ and Ca+ of your intracellular pipette? Increasing Mg+ and Ca+ concentration may help with gigaseal formation:
Ionic Requirements for Membrane-Glass Adhesion and Giga Seal Formation in Patch-Clamp Recording
If this is your first time patch clamping neurons, it may be easier to start out young or even embryonic DRGs which should be much easier to patch and would allow to you see if the technique itself if the problem or perhaps the age of the DRGs.