Could someone please give me the recipe for the internal and external solution to measure sodium and potassium currents in cardiomyocytes, whole-cell configuration and voltage-clamp mode?
I have stable gigaseals, nevetheless, I have used MANY solutions for two years but have not been able to get the whole-cell configuration to hold for more than 5 minutes.
I think it is due to the osmolarity of our solutions BUT we do not have an osmometer in our laboratory and although I control other variables (pH, bath resistance, seals, etc), I have not been able to obtain stable records.
Dear Jessica,
It is very difficult to identify your problem from a “theoretical” frame. Regarding the solutions, we performed patch clamp recordings for decades also with many different solutions that were also different to the solutions used by other groups and, we had not your problem of instability. You have also used many solutions with the same problem and so, provably the problem is not the solution.
However, I assume that your external solutions have Calcium. The absence of Ca2+ in the external solution makes difficult to seal the membrane with the glass.
The second possibility I guess is the glass used for the patch pipettes. The best is hard glass, like thin wall borosilicate tubing. Other types of glass, like aluminium silicate or other soft glasses makes seals easily, but do not hold for more than few minutes.
Finally, the most common reason for seal loss is an air loss in the suction system. All the tubing connecting your pipette with the suction system must be tightly sealed. A small, unappreciable puncture or air loss in the system makes the negative pressure to disappear and the seal to fail. If this air loss is small enough, the seal loss could last some minutes.
I hope you can find a solution to your problem. Good luck,
Oscar
There’s a few mechanical things to check. Sorry. You probably know these, but one has to check.
* Make sure your air table is floating properly. Not resting on the table legs, but also not pegged at maximum height.
* Check the stability of your manipulator. Make sure it isn’t drifting.
* Tape down your pipette holder suction line. If that tube gets yanked at all, it can move the electrode and disrupt the patch.
* Make sure the line is clean, with no fluid in it.
* Avoid touching the table or manipulators once you break in.
* I suppose you already have tried changing how you seal (don’t use too much positive pressure when you go down on the cell) (get a dimple, but don’t shove too deep) (pull back on the electrode once you break in).
If you are worried about your osmolarity, find someone with an osmometer. You don’t have to check it often. It is something that should be adjusted. Recipes often assume you have one, and theoretical estimates of osmolarity are usually off. On the other hand, people vary in what osmolarity they prefer, so 5 or 10 mOsm probably doesn’t matter.
Good luck!
I really appreciate that you have been able to answer my question. Last week we changed the micromanipulator and have noticed an impressive improvement in our stability! We had a really big drift. Our solutions have been working well.
Thank you so much!
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