recording chamber

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zhangxu04
zhangxu04's picture
recording chamber

hi,
 
I used the RC-24E to perform the patch clamp, which works very well for several months. However, I meet some very depressing problem. The seal was deteriorating significantly after achieving the whole cell recording mode and the leaking current is increasing with time going even increases every sweep. I have my system and find out this is directly related to recording chamber. Now I have to clean the chamber totally and change the matched coverslip everyday so that the seal will be stable as long as more than one hour.
 
My chamber is RC-24E, coverslip cat: 64-0707 and I used the Dow corning III and my clean protocol for chamber is just following the recommended method in Warner web using the TSP. so could you please give some suggestion

thanks a lot

The FFM
The FFM's picture
It seems that you are

It seems that you are effectively troubleshooting this yourself so far.

Just make sure you chamber is always clear of salt and drug from any previous experiment.  Are you using a drug that might be absorbed into the chamber plastic during your experiments?

Check that the osmolarities of your pipette and bath solutions are correct every day

Check that the cells that you are patching are as healthy as possible during thecell culture befor you patch them.  Have you tried allowing the cells to equlibrate for 15-20 mins in the bath solution before you put them in the chamber and patch them?  Or is the Osmolarity of the bath solution matched to that of the culture media, so that they are not experiencing any osmotic shock when you change solutions?

zhangxu04
zhangxu04's picture
thanks a lot.

thanks a lot.

in fact I feel the problem is related the Osmolarity but not very sure as I can not measure it.  it seem that the Osmolarity in the chamber will decrease which is  almost unbelievable. for example I incrase the Osmolarity  of bath solution and it can work for some day then it will be like former situation. even some times the seal can be back to G seal when perfused with compound sulution, which containning 0.1% DMSO .

please give me some advise.

thanks