Hello. I moved to a new lab. My IP solution, which I used at previous labs, stopped working. I can not hold a cell for more than 5-7 minutes, then leak current increases dramatically and I lose the cell. Forced resealing shows that the seal between the cell's membrane and pipette remained good. No significant electrode drift was observed. What may be going wrong? IP solution (mM): K-gluconate - 105, KCl - 30, CaCl2 - 0.5, MgCl2 - 2.4, HEPES - 10, EGTA - 5, ATP-5, Na-GTP - 0.4, pH - 7.3, 340 mOsm. Bath solution: NCl - 145, KCl- 5, CaCl2 - 2, MgCl2 - 2, HEPES - 10, Glucose - 10, pH - 7.3, 330 mOsm. IP solution is held on the ice during the experiments, and the temperature of the bath solution in the chamber is maintained at around 37C. Thank you.
PS I tried different ATP salts for the IP solution: disodium, dipotassium, and magnesium. The results were the same. EGTA: I use acid, not sodium salt.
Hi Viacheslav!
From my experience, one usually tries to have a lower osmolarity and pH in the intracellular solution compared to the bath solution, 340 is already quite high. So I would aim for 315 - 320 mOsm and pH 7.1 or something like that, it should usually increase the patch success. A bit more GTP could also help.
Are you patching different cells now than you did before? Different cells might have different (ionic) needs, so playing around a bit with slicing parameters/concentrations etc could help, maybe there is already something in the literature that could help (or your lab might have some knowledge on the cell type.)
Good luck! :)
As the previous person I think your osm is too high - we have extracellular 308 and intracellular 295 pH outside 7.4 inside 7.2.
Dear Victoria K. Switacz, Thank you very much! I previously worked with rats' DRG neurons, but now I'm working with mice'.
If others in the lab are also doing electrophysiology, try borrowing a little of their internal, and see if it makes a difference.
The behavior you describe is also consistent with poor cell health. Maybe the isolation procedure needs refining. Or try a DRG prep from a lab mate, and see if it makes a difference.
Good luck.
-Matthew
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