Low membrane resistance

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Recorder
Recorder's picture
Low membrane resistance

Hello, thanks for reading my question.

I'm doing whole cell patch clamp recording with cultured hippocampal neuron. For the neuronal culture, I'm using co-culture system with astrocyte. I cultivate the neuron on top of the astrocyte layer. The neurons looks good and healthy.

However, I'm suffering serious problem. When I go into the whole cell, after the giga seal formation, usually the membrane resistance is quite low, mostly below 80 Mohm, sometimes even below 50 Mohm. Although I got a whole cell with good membrane resistance rarely, the membrane resistance is usually drop to very low resistance quickly, typically within 10 min. For the purpose of my research, I have to maintain the whole cell configuration quite long time, like 30 min.

Increase of access resistance during the experiment is quite usual, as well.

Is there anyone who can give me any advice to solve these problems?

Thank you very much.

Just for your infomation, I'm using NaCl based external solution with bicarbonate buffer system. And for the internal solution, I'm using K-methylsulfate as a main component, but I also tested K-gluconate. The experiment was performed under 35 degree Celcius perfusion chamber.

The FFM
The FFM's picture
Have you tried obtaining the

Have you tried obtaining the seal and going whole cell at room temp and then increasing the temp to 35 degrees once the seal is stable?

the membrane is much more fluid at 35 degrees so it is really hard to maintain a good seal for longer than 15 mins. Is it absolutely necessary to be patching at 35 degrees for your applications or can you infer function from room temp expts?

Recorder
Recorder's picture
Thank you very much for your

Thank you very much for your advice.

In fact, mimicking natural envirenment is quite important for my purpose. But, maintaing stable recording is essential anyway.. If the low temperature is work... well I don't have other choice, I think.

I'll test the low temp. Thank you very much.