creating a good seal in acutely isolated neurons

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Papillon_Cat
Papillon_Cat's picture
creating a good seal in acutely isolated neurons

Hi all,

I am having a lot of difficulty lately patching acutely isolated neurons from CA1 region of hippocampus. I have no trouble with sealing and breaking through, the problem arises when I lift the cell (after breakthrough) off the plate. Shortly after lifting the cell into the stream of solution, the cell falls off the electrode. I wonder if the problem has to do with the seal itself, the osmolarity of my solutions, CsF in my ICF....

The ECF of my solutions is usually 325-300 mOsm. The ICF is usually between 295-300 mOsm.

Please let me know if you have any suggestions.

Thanks,
Catherine.

The FFM
The FFM's picture
 I always found I had more

 I always found I had more success if I patched and got the Giga seal, then lifted the cell, and only after the cell was successfully lifted did I go whole cell and put it in the solution stream.

Gruub
Gruub's picture
Also check the speed of your

Also check the speed of your stream. I got similar problem with freshly digested arterial SMCs
I use to get the gigaseal while the cell is on the coverslip and lift the cell afterward.
Before I went to wholecell, the cell was sometime washed away once in the stream (1ml/min)
I worked around this problem by widening sligthly the diameter of the exit (to maintain the debit but reduce the speed of exit) of my test solution stream and reducing the debit to 0.3ml/min

To allow quick test perfusion change I also used perfusionm tip pulled from teta tubing (or tri tetra etc up to hepta) which allow change of solution without dead space

link here