Dear Alice: Indeed, the electrophysiological recordings on cultured astrocytes were not be easy. Our paper might be useful for your recordings. One could be better to check the function of vibration-free table, if the recording pipette was often broken during measurement.

No. There seems to be no other possibility except to be patient, use a very stable manipulator, good vibration isolation and take care to move focus between the ceel and electrode when aproaching. I am adjoining a text with brief methodology.

So, under whole-cell recordings, one might think that the electrode appeared to be broken down. It is possible that ionic currents inherently in astrocytes appeared during the recording. One might need to known where the value of reversal potential under each experimental condition used.

Thank you for your answers.

Hello Alice,

1/ If you're breaking your pipettes more often than usual, then it could only mean a mechanical issue. I may be wrong but your approach to the Astrocyte is probably too quick. To solve that, you have to get used to very very slowly and gentle approach of your cell. If you happen to have a Sutter ROE-200, you can set the fine movement to the lowest possible value (I believe, it's 9 for that instrument). That might help (even though Sutter recommend 5 to 6 in the manual which is good for other cells).

2/ When your pipette does not break, you still don't get a seal! Try the follwing: (i) I am sure, you do already use this, but I will mention it anyways: Have a small difference in Osmolality between your external and internal media. Your bath (305-315 mOsm/Kg) should be 20 to 30 mOsm/Kg higher than your internal (280-285 mOsm/Kg) (ii) try to seal in high Calcium and once you get your seal, go back to your normal external solution. Of course, do this in combination with the first suggestion.

I have no experience patching Astrocytes although I did patch all kind of cells (even from plants and yeast). Some cells require longer time to establish a Gigaseal than others. So, when you are sucking your cell, do it in small steps and watch your monitor. when you get up to 100 MOhms or so, go to Whole Cell mode and set your Holding Voltage to -30 mV (or +30 mV). Toggle between these values and Zero mV and watch your Gigaseal form.

With that, I Wish you happy and successful patching - Fouad L-C

Hi Alice,

I'm in the same trouble now...I'm easily reaching the gigaohm seal with a KGluconate intracellular solution. But it appears quite impossible to break the patch and achieve the WC-configuration..it is more likely to break the seal.

Have you improved your methods...any suggestions? (Rp 5MOhm)

Marcelo, think about using perforared patch?

Hi Enrique...very thanks. This is agood Ideai was thinking on to...but since I also need to track the cell shape.. does neurobiotin/lucifer yellow pass touch amphotericin pores?

Very thanks

I don´t know about permeability of Neurobiotin/Lucifer yellow trough nistatin, amphotericin or gramicidyn pores, but consider that cAMP cannot pass through. So comparing weigths may lead a hint about permeation.