Abrupt Patch Clamp Problems

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jsvick
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Abrupt Patch Clamp Problems

Hi everyone,

I've been doing perforated and whole-cell patch clamp in my lab with isolated vomeronasal sensory neurons for about 3 years now.  I've had problems in the past, but these have always been solved by new solutions, new filters, changing puller settings for better electrode tips, better cell isolation, etc.

Recently and very suddenly, I've been having problems where my seals will not form beyond 400mOhms (700mOhms if I'm lucky).  I've already put on a new electrode holder, new O-rings, and new tubing/syringe to no avail.  I know it is not the cells, as the problem persists even if my labmates isolate cells for me with their solutions when they have been getting good cells/gigaseals.  The odd thing is that there will be a day every two weeks or so where I can get great seals and very stable whole-cell configurations.

Regardless of if the seals stabilize at 400mOhms or are > 1GOhm, it takes much longer to seal then it used to (about 20 minutes as opposed to 5 or 10 minutes).  Another very strange thing is that when switching from seal test to membrane test in Clampex where the cell is voltage-clamped at -60mV, I always see a drop in the membrane resistance (for example, from 40mOhms to 30mOhms).  This has never happened before; even with cells that in the end would never seal STILL have at least some increase in the membrane resistance when switching to membrane test.  I have checked the model cell on my headstage, and all of the electrical parameters are normal.

I'm beginning to suspect that I may have electrode drift, which I will need to check, but doses anyone have other suggestions?  Thank you for reading.

Simon8
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Hi.

Hi.

Did you check if your pipettes are the same? Same resitance/shape? Maybe your puller needs a new filament or the program was changed?

jsvick
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Hi Simon8,

Hi Simon8,

I have double-checked the puller's settings, running a new ramp test etc., and the ramp hasn't shifted much.  Normally if our filament burns out, cracks, or otherwise becomes compromised, we see a drastic change in the ramp and our tip sizes/shapes become unreliable.  This hasn't happened, and my labmate has no problem pulling good tips and getting good seals.

My major concern is with negative holding potentials seemingly disrupting my seals, which always occurs when I switch from seal test (test pulse of 2mV) to membrane test (holding potential of -60mV).  This used to never occur.

Simon8
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Ok, where do you set your

Ok, where do you set your holding potenitals? via the software or the amplifier itself?
Did you check the IN and OUT settings for Seal- and Membrane-Test?

jsvick
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We set the holding potentials

We set the holding potentials with the software.  Can you be a little more specific for the IN and OUT settings for seal test and membrane test?  What should I be looking for, specifically?

Simon8
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Clampex (wich I suppose you

Clampex (wich I suppose you use?) can be quite confusing with all these Inputs and Ouputs and different Signals you can set up.
Somewhere there is a setup page for Seal Test and Membrane Test. Check there, if the right signal is selected. You also should re-check if all the settings for THIS signal are OK (I think its in the "Lab Bench"), expecially the scaling-factors.

My idea (and nothing more) is the folowing:
You Seal Test is properly set up. But somehow you Membrane Test has not the right settings, so the apllied potential might be wrong, hence when you turn in on, your clamp messes things up and you might loose the seal.
However, this is not very probable, because I guess you did not change anything.

Another idea that comes to my mind. Do you use the same (really the same, not only the same type, but the same lot etc...) perforating agent as your collegues? Maybe it is not good anymore?

jsvick
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I'm sorry to necro this

I'm sorry to necro this thread but I solved the problem a few months ago and felt I should update.  It turns out that there was an issue with my Multiclamp 700A.  When I switched to a new one, voila, seals were excellent and whole-cells were stable.  Thanks for all of your suggestions!