Does anyone have insight as to whether it's possible for K-internal to go bad?
We are performing whole-cell patch-clamp recordings. For the past few months we have been having issues with cells dying shortly after forming a gigaohm seal and breaking in. I can only hold a cell for 10 minutes or so. We have checked pipette drift, the pipette isn't drifting. Our slice quality is fantastic. The only other factor we can think of is internal solution. All of our internal is made, dated, placed in hundreds of small containers, and only one or two containers are thawed on the day it's time to patch.
At this point I've patched onto hundreds of neurons with this problem, statistically at least one should have survived past 10 minutes but none have.
Is it possible for K-internal to go bad?
Dear Ian,
There's always the possibility of you internal to go bad. However, it'd take a good amount of time, especially if stored properly. I'd suggest trying a different internal. Ask a friend maybe from another lab if they can give you a vial of theirs. That's the best control you can have. Some more topics to think about:
- water quality. I had issues with less than ideal ultra pure water in the past.
- osmolarity delta. Check these properties for both internal and external. See if they're in a healthy state (up to 10 mOsm would be ideal).
- stocks. Do you use 10x stocks to make the external? Is it a new batch? Do you trust it?
Good luck!
Andre
This is a tough one. Andre Dagostin had some good suggestions.
On the question of the longevity of internal solutions, I'll just add one data point: I typically keep aliquoted internal solutions in the freezer for ~4 months. I've been doing that for 20+ years and I've never had a problem.
By all means, check the osmolarity and pH -- and you might as well make up new internal (why not?) -- but I doubt that's the problem.
I make new solution every 3 months, aliquot it and store it in the -80.
Besides filtering, cheking osmolarity and ion composition of your patch solution, you may try perforation with amphotericin as an option for WCR.
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