Lose the seal when holding the membrane at -60mV

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patchclamper
patchclamper's picture
Lose the seal when holding the membrane at -60mV

Hi everyone,

After getting a seal, I hold the membrane at -60mV then break in. There was no problem with it before, however recently, when I hold the membrane at negative potential, I lost the seal almost everytime (some times go to whole cell directly). Do you guys have any idea what are the potential causes for this?
I am thinking of osm of solutions, negative pressure, problem with amplifier's v-clamp (Axopatch 200B)..

Any ideas?

cellpatcher
cellpatcher's picture
You don't lose the seal or go

You don't lose the seal or go whole cell directly until you hyperpolarize? 

patchclamper
patchclamper's picture
No, The seal is very stable,

No, The seal is very stable, until I hyperpolarize the cell...

Yesterday I found that my fast electrode capacitance compensation is off, it seems when I hyperpolarize the cell the compensation circuits is generating some osllilation which will potentially damage the seal. Today I will do a recording again and see whether that is the case.

Frere Jacques
Frere Jacques's picture
Hi patchclamper!

Hi patchclamper!

We've been having the same problem as you lately: we get a nice seal, then start hyperpolarizing to -60mV and at about  -20 or -40mV we seem to lose the seal as the square pulse is no longer flat. However A) I've noticed that most times the pipette is still strongly attached to the cell and I rip the cell by pulling the pipette away, and B) this problem went away for a few weeks without me doing anything special and just came back suddenly (no soluition, or temperatuer, or pH, or osmolarity change)!

So I was wondering: did you fix this problem and how? Anyone else has a suggestion???

Thanks! 

patchclamper
patchclamper's picture
Hi Frere,

Hi Frere,

I am still having this problem now, similarly, when I hyperpolarized the seal about -40mV, the input resistance decreased from 2 GOhm to about 15 MOhm.

Whether the decreasing of input R is due to losing the attachment of membrane to the glass or due to going to whole cell directly is hard to say, often even the seal is broken, you  can still pull out the electrode with the membrane  going with it.

Potential cause for this  could be that the current injecting to hold the cell is wrong, eg. forget to  zero the pippette potential, gain of amplifier setting, data aquisition software gain setting and offset voltage, and other current source in the bath (eg, stimulator leaky current).

Still haven't figured out yet, and to avoid losing the seal,  I break the seal by suction at -30mV and after getting whole cell, i further hold the cell to -60mV. However, It's still important to know why this happens....

Let me know if you have new ideas.

Thanks~

A similar topic:
http://www.scientistsolutions.com/t10483-acute+hippocampal+slices.html

patchclamper
patchclamper's picture
 Still have the same problem,

 Still have the same problem, now I changed my amplifier to 700B, and everything works fine...

But still have no idea why my old 200B will cause this "hyperpolarization induced seal lose"....Probably something wrong with the gain...