Hello:
I am doing patch clamp in HEK cells. Very often I have problem with not having gigaseal at all. In case I succeed with giga seal the patches are not stable at all. I am using the foloowing solutions
extracellular solution in mM: NaCl 135, KCl 5, HEPES 10, Glucose 10, MgCl2 1, Cacl2 1
intracellular solution in mM: KCl 140, NaCl 4, MgATP 2, HEPES 10, EGTA 5, Cacl2 2,2
I will appreciate if you have suggestions and comments.
Thanks a lot in advance.
Hi there,
I also had problems with unstable and leaky seals.
A couple of weeks ago I switched my intracellular solution from (mM):-
150 KCl, 1 CaCl2, 10 HEPES, 1 MgCl2, 4 K2-ATP and 0.3 Na-GTP.
to (mM):-
140 K D-gluconate, 10 HEPES, 1 MgCl2, 2 K2-ATP and 0.1 Na-GTP.
As you can see, no Ca2+ and much less Cl-. About the lack of Ca2+, a postdoc told me it wasn't a good thing, as all cells need intracellular Ca2+. About having much less Cl-, one of my PI's said something about reversal of Cl- potentials..... but I don't entirely understand what that means. My supervisor said that I still have the main cation (K+), so the change in solutions shouldn't void all my previous work. For now I'm just seeing how it goes, but, by switching intracellular solution I have noticed that I am more likely to form a giga seal and the seal itself is more stable.
I do whole cell patching in current clamp in hippocampal pyramidal neurons from acute brain slices, so I'm not sure how relevant this is to you, but hopefully it's useful.
Hi! thanks a lot for your replay.
I am using different extracellular and intracellular solutions at the moment to find out the reason. Regarding to your new solution: from my experience I know that with K-gluconate based intracellular solutions is easier to patch. Before I worked with brain slices and never had problems with patching.
Best!