problem with pore formation

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T9
T9's picture
problem with pore formation

Hallo!

I do perforated patch clamp with gramicidin on tout cardiomyocytes. I have next problem. When I got gigaohm seal, the pipette compensation is well done and I wait pore formation. After 10 minutes, the trace looks like pores are formed but suddenly at about I=-200, the I jumps to -3000 and trace changes also. Can you help me what could be a problem?

The FFM
The FFM's picture
It sounds like your patch

It sounds like your patch seal gets blown and/or the cell gets super leaky.

What is the seal resistance after the holding current jumps to -3 nA?  

What concentration of gramicidin are you using?  

do you sonicate your pipette solution immediately before you backfill the pipette EVERY time you load a new pipette - you should.

Have you balanced the osmolarities of the extracellular and pipette solutions?

Are you keeping the cell under constant fast perfusion while you obtain the seal?  If there is residual gramicidin in the vicinity of the cells that came from the tip of the pipette before you obtained the giga seal then all the cells in the area will become perforated by the ionophore.

I also refer you to Chapter 8 of this book which may help you troubleshoot your perforated patch experiments

Perforated Patch-Clamp Techniques

Book Series
Neuromethods

ISSN
0893-2336 (Print) 1940-6045 (Online)

Volume
Volume 38

Book
Patch-Clamp Analysis

Edition
Second Edition

Publisher
Humana Press

DOI
10.1007/978-1-59745-492-6

Copyright
2007

ISBN
978-1-58829-705-1 (Print) 978-1-59745-492-6 (Online)

DOI
10.1007/978-1-59745-492-6_8

Pages
253-293

Subject Collection
Biomedical and Life Sciences

SpringerLink Date
Saturday, March 15, 2008

T9
T9's picture
The seal resistance is about

The seal resistance is about 200Mohm. Gramicidin is 0.1mg/ml. I do not do any sonication. Is it necessary?
I have no idea about my solution osmolarity and do not know how to it.What osmolarity is best fr solutions? I do not keep the perfusion all the time, cells drift in solution otherwise. No it did not come from tip, because if it happens cell immediately dies. But in my turn it does not die.

The FFM
The FFM's picture
With gramicidin concs of 0

With gramicidin concs of 0.1mg/ml  I would expect that you will be seeing spontaneous rupturing of the membrane patch for most of your recordings.

Try reducing your gramicidin to 20-25 µg/mL  (1/4 of what you are presently using).  And even then you must prefill the very tip of the electrode with a little antibiotic free solution to avoid interference of the antibiotic with seal formation.  If you don't do this and your are not perfusing the extracellular solution over the cell while you are attempting the patch, a small quantity of antibiotic WILL leak out the tip of your pipette and over time perforate the whole cell and not just the patch.

However that may still not be enough.  Then you will have to reduce the gramicidin conc down to 2-5 µg/ml.  It make take up to 30 minutes to get a fully perforated patch after you have achieved a Giga seal, but you won't need to prefill the tip with antibiotic free pipette soln and you should see much less spontaneous rupturing.

Frequent sonication of the gramicidin is usually necessary to ensure that the antibiotic is fully in solution and will maximize how quickly  the ionophore works in the patch.

RE: osmolarity.

Most physiological solutions are between 296-330 mOsm

You can check the osmolarity of your solutions using a vapor pressure osmometer - these devices are made by companies such a Wescor.  Pipette solutions should normally have an osmolarity about 5% less than the extracellular solution.

E.g pipette soln 295 mOsm
       Extracellular solution 310 mOsm

you can adjust the osmolarity of your solutions by adding an appropriate amount of sucrose.  If the difference between the osmolarities of your solutions is significantly more than 5% you may well have problems keeping a good seal.

T9
T9's picture
Oh, Thank you for this

Oh, Thank you for this precious information I will today control the osmolarities and try today your advices about conc reducing and superfusion. Actually I have been using previously the amphotericin B instead of gramicidin I used same cocn and I got good gigaseals and recordings also. I laso have not any problem with compensations. This is why I used same conc for gramicidin and all the articles suggest same conc.