Hello
I tried to make perforated patch recordings of cultured neurons with amphotericin B. With only a few cells I could get R > 1 GOhm. Usually resistance rises up to 200-600 MOhm and then starts falling. When it falls to 100-200 MOhm capacitance transient slowly develops (Rs 40-80 MOhm) and voltage can be measured in current clamp (usually around -40 mV while "standard" patch-clamp gives -70..-60 mV).
I wonder what happens in these cells? Why I don't get gigaseal as usuall? I tried amphotericin B concentrations between 100 ug/ml and 150 ng/ml (although no cell access can be established with 150 ng/ml), tip filling from 1 second to 1 minute, pipette resistances from 2 to 7 MOhm, both applying and not applying positive pressure while approaching the cell.
You advice is greatly appreciated!
Hi Misha,
It sounds like the amphotericin starts acting before you have time to get a giga seal. I am not sure how you fill your patch pipette but you may want to fill the tip of the pipette with an internal solution without amphotericin and back fill with the solution containing amphotericin - that way you have time to get a giga seal before the amphotericin get into contact with the cell membrane.
Hi Misha,
It sounds like the amphotericin starts acting before you have time to get a giga seal. I am not sure how you fill your patch pipette but you may want to fill the tip of the pipette with an internal solution without amphotericin and back fill with the solution containing amphotericin - that way you have time to get a giga seal before the amphotericin get into contact with the cell membrane.
Hi Norbert
this is exactly what I was doing: filling the tip with amphotericin-FREE solution (by immersing the tip into solution for several seconds, up to 60 seconds in some cases) and then backfilling with amphotericin-CONTAINING solution.
Hi Misha,
I worked with Amphotericin before and I used the same method as you did. I also used another way, by having only amphotericin in the pipette solution but keeping the cells under perfusion, washing out any amphotericin that could harm the cells before reaching the seal. This way worked fine for me, maybe you could give it a try?
Good luck!
Hi Vincenzo Mastrolia !
did you work with cultured neurons or with slices? What was the amphotericin B concentration and what was the pipette resistance?
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