While testing the osmolarity of our solutions, we noticed our frozen and aliquoted K-internal solutions have an osmolarity ranging from 270-290, while our external aCSF has an osmolarity ranging from 310-314. Is this difference enough to cause cells to shrivel and die mid recording? Thank you for your response, its much appreciated.
30 mOsm/kg is the threshold. A gradient of 30 mOsm/kg or more produces significant shifts of intracellular water and cell damage. I suggest you keep the osmolarity of our internal solution at around 10 mOsm below the aCSF. You will have (mostly) good results with getting seals. Leave the aCSF osmolarity as is, and increase the osmolarity of your internal solution.
Yes, this difference is too big. Aim for a difference not exceeding 5-10 mOsm/kg. Are you mixing the solution before use? This 20 mOsm/kg range is too much. You could add some sucrose to the internal solution, as a quick fix, assuming you haven't left anything out (HEPES? EGTA?). For the ACSF you could replace part of the sulphate (if you have that in) with chloride salts and maybe decrease NaCl a bit. (Check the Nernst potentials to make sure you get what you need before adjusting salts!)
Dear Ian Morley ,
Echoing my colleagues above: a delta greater than 10 mOsm/Kg is not adequate. In my experience, it is not a problem if the internal or external is more concentrated (it depends on the preparation) as long as this rule is not broken. And since your internal seems low, I'd review its formulation.
Good luck,
Andre
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