patch clamp

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vard
vard's picture
patch clamp

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.

c.hill
c.hill's picture
Hi there,

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.

vard
vard's picture
Hi! thanks a lot for your

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!