Aspirating nucleus during seal formation

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Simon8
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Aspirating nucleus during seal formation

Hi!

When trying to do whole-cell recording in dissociated cortical neurons, I am very often not able to get a gigaseal, because before the seal is formed, I somehow snag the nucleus. I read that this is called a "nucelated patch". But I do not want it, and I have no idea what I can change to avoid this.

I approach the cell without pressure and after an approx. 30-50% increase of the resistance (pipette has around 5 MOhm), I apply some suction to increase the seal. On the other hand, sometimes I am able to open the cell, but I can not tell what the difference in my approach was.

Anybody else has this problems, or does anyone know what goes wrong here?

Simon8
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In the meantime I was able to

In the meantime I was able to increase my success rate, but I cannot give a reason. Looks like I just had to train a little more.
The problem with the nucleus occurs, when sealing takes a lot of time, and plenty of suction has to be applied.

I will keep you updated if find out more.

Edit: I think it is the quality of the cells that matters here. Recently I again had the problem, and I think my culture is not so healthy (I need older ones, than are used by other people in our lab). I will see if younger neurons are better.