How often do you get new salts? What salts do you get "fresh" most frequently? All help is greatly appreciated. Thank you to everyone who reads or responds, hope you all have a fantastic day.
I am a Graduate Student at the University of Wisconsin Milwaukee. Our most recent electrophysiology experiment was going fantastic for about 3 weeks. Suddenly, every slice was very poor quality, and every cell we patched onto died within about two minutes. Even if you don't have an Electrophysiology background, all advice and input is appreciated. It's been two weeks since this problem first arose. We didn't alter any procedures, everything has been held constant. We don't know what could be causing the sudden change in slice quality.
So far we have three theories: DDH20 filter needs replacing, our glassware has somehow become contaminated, or our salts have gone bad.
The DDH2O water resistance reads about 14.8MOhms. We changed the filter about a year ago. From what I have read, DDH2O should be between 14 and 18 MOhms to ensure slice quality, so we think our water is fine.
Our glassware washing procedure is 3 rinses of tap water, 3 rinses of deionized water, and 1 rinse of DDH2O. We don't know how else we can safely clean the glassware for slice preparation. But we don't think this is the issue.
Our main theory is the salts have gone bad. Our lab is on the 4th floor, and it is usually quite warm and humid on our floor during the summer. Most of opened salt bottles we use were first opened between 2 and 7 years ago. (for example, our bottle of sodium phosphate monobasic monohydrate and our potassium chloride were both opened 6 years ago. Our magnesium chloride and sodium chloride were both opened 2 years ago)
We think with repeated opening of the salt bottles causes debris/water from the atmosphere to leach into the salts, resulting in a change in our solutions and causing the cell death we have been observing. Is this a real possibility? How often do you get new salts? What salts do you get "fresh" most frequently?
We checked the pH, the pH of our solutions is within .1 of what it should be. All help is greatly appreciated. Thank you to everyone who reads or responds, hope you all have a fantastic day.
The only way these salts would change would be if they absorbed water from the air. This would result in your salt solutions being less concentrated than expected. Some of the salts you mentioned are hygroscopic, but unless you notice that they have become highly caked, or that there is liquid in the bottles, the amount of water absorbed is probably not very much.
If pH and osmalarity are what you expect, the chemicals should be fine. Make sure the incubation and recording chambers (including tubing) are clean, and check the ACSF flow. If the cells are dying a couple of minutes after opening, maybe the issue is with the intracellular solution or vibration/movement.
These salts cost very little, and I would simply throw them out and buy new ones. If the issue goes away, great, If not, then you have narrowed down the options very quickly with little cost. You could also buy a bottle of sterile water to rule out some issue with your production system (I think you can have contaminants that do not affect the resistivity).
Hi! Every (electro)physiologist feels for you; nothing is more annoying than experiments suddenly stopping working...
A wrong slicing angle could also be a reason for poor-er slice quality. Check your blade holder, possibly someone changed it, and it may now be too steep or too shallow, causing additional stress to your slices. And I would, as advised before, slowly replace stuff that can be easily replaced one by one to rule out the problem: Salts, tubing, water, slicing/storing chamber etc. Good luck! :)
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