Cannot break into the cells

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vilsy
vilsy's picture
Cannot break into the cells

Hi Guys,
 
I am doing acute thalamic slices and am trying to patch cells. I am able to get a gigaohm seal easily, but then am not able to break into the cells to go intracellular. Applying brief pulses of suction or even zapping using the Zap funtion of the MultiClamp 700B is not helping me. I am trying to think of everything that i can be doing wrongm but cant think of anything.
Did anyone have the same problem? If yes, can u suggest me something that I can do (as in change the protocol or solutions a little) to break into the cells?
 
Thanks,
Vilay

cuizy2009
cuizy2009's picture
I have the same puzzle in my

I have the same puzzle in my experiments.
I use the primary cultured hippicampal cell of  rat.
I guess the most possible problem might be the cell state, I tried many modification, but no much improvement.
This bother me for a long time, I need some help,
Anyone can help us??????

The FFM
The FFM's picture
 did you ever try sucking and

 did you ever try sucking and zapping at the same time?

neuro
neuro's picture
Have you overcome your

Have you overcome your problem with rupturing cell membranes?  I have been whole cell vclamping pyramidal neurons in the mouse auditory cortex for a couple years now... while other publications recommended a recording solution of 285 - 295 mOsm (before adding divalent ions), I found the cells looked a bit swollen at this tonicity.  I found the neurons became more typically shaped (triangular) at slightly higher osmolarities of 300 - 305 mOsm, and I found that I had a much greater success rate of breaking into the cells.  Finally, my electrode resistance needs to be between 4 and 5 mohm.  Any lower and forming a seal is difficult; any higher and rupturing the membrane becomes more problematic.

Lostbrain
Lostbrain's picture
I also think this is to do

I also think this is to do with the slice quality. Does the slice look sticky or too rigidwhen you insert a pipette? Did you work previously with other slices in which the problem did not happen? Improving slice quality is a pain and might take ages. I would recommend you to make a list of all possible parameters during the slicing process and, every day, make a note on those. Relate that with slice quality and change things gradually to see if there is improvement. I did that very systematically once I had terrible problems and worked pretty well after a few weeks.

Addtionally I would recommend to use a syringe instead your mouth and give very short and gradually more intense suctions. If after one or two suctions does not work, then apply zap and repeat. If it does not work, increase the zap amplitude, and so on. Don't get impatient with a cell and don't start sucking too strong, and keep doing that progressively. Perhaps will work.

Another possibility is that you think you have formed a proper seal, but what actually happened is that you blocked your pipette. Can you see single channels in cell attached? Is seal formation smooth or do you have to suck quite a bit?

Cheers

tbonds
tbonds's picture
 How long do you wait from

 How long do you wait from the time you form the Gohm seal to the time you go whole cell?  If you wait too long, the membrane itself begins to fold in on itself within the tip and it then becomes impossible to break-in.