Hello,
I wish to perform patch clamp recordings on confluent human iPSCs derived RPE cells.
The problem is that when they gather to form the epithelium these cells are linked to vicinal ones via gap junctions. So it is a nightmare to try to impose a voltage to a specific cell because of ions continuously flowing between neighboring cells.
Applying a wide spectrum gap junction blocker such as carbenoxolone in the perfusion solution would block them irreversibly in the whole epithelium and I am not sure that it would be fine for the cells. I need to keep them as healthy and functional as possible during the experiment to get a maximum of correct data from each RPE.
My idea is to put a specific blocker in the pipette medium in order to block gap junction only in the patched cell. This would allow me to patch several cells in the same RPE and avoid possible deleterious effects of a massive and irreversible gap junctions inhibition.
I haven't seen such an approach in the literature, usually blockers are applied via the perfusing solution.
Does anyone know a paper in which such an approach is used ?
All constructive comments are welcome.
Thank you in advance.
Patrice
Dear Patrice,
It is a difficult situation. Have you tried to enzymatically dissociate the RPE cells prior to recording? That may be worthwhile trying to do and should be relatively easy.
Another way may be to include carbenoxolone in extracellular solution in a small tip pipette and gently apply the drug on to the cell of interest for a few minutes, then go back to the same cell with your normal patch electrode and seal and clamp. If you have a second holder (two-electrode set-up), then you can apply carbonoxolene with one microelectrode and then patch-clamp with the other microelectrode. You may also be able to angle the flow of carbonoxolene in a direction relative to the the flow of your bath solution that will minimize the effect on other cells.
Best wishes,
Refik
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