Hello everyone,
I'm having difficulty keeping stable recordings from my E18 cortical cultures. I'm trying to record mEPSCs and I'm voltage clamping at -70 mV. My setup is an Zeiss axiovert 25 inverted microscope, an Axopatch 200B amp, a Axon CV203BU headstage, and a Sutter MP-225 manipulator. I'm using King Precision Glass KG-33 electrodes with filament: 1.12 ID, 1.50 OD, and pulling them at 3-5 MOhm. I can make a gigaohm seal without issue usually, but once I break in, there is so much leak and my holding current is so unstable that mEPSC events become almost completely undetectable, and I usually lose the seal entirely within <1 minute. When I make a gigaohm seal, there also seems to be drift in the Y-axis that i think may be one of the contributing factors for the instability of my seals. However, I have tried everything I can think of to get rid of the drift, but to no avail. Could this be an issue with the cells themselves? Patching cultured neurons is e a very common occurrence, so I'm not sure why I'm having such a difficulty here.
Any suggestions will be much appreciated. Thank for you taking the time to read my question!!
My five cents.
First of all, I would make sure that you're not too "rough" when you're breaking the membrane, after you form the gigaseal. I use mouth to break the membrane. Also, check the osmolarity of your internal/external solutions.
If you have a drift, after forming a gigaseal, I would suggest to re-chloride your ground electrode (I don't know if you're using the AgCl ground electrode/pellet or not) TOGETHER with your silver wire from pippette holder.