Hi, colleges, thank you for your reply. I update my descriptions in my attachment. So sorry for the miswrite of water. It is actually ACSF.
I have two questions.
Frist, when the pipette into the ACSF, it is overload and I need to offset several times.
Second, the Ra is increase during the time, how to deal with it.
I have checked the osmolarity. ACSF is 294 mOsm/kg. And Cs- ICS is 286 mOsm/kg.
The old version of question:
I put the recording electrode into water (actually ACSF) and test the pipette resistance. When offset the pipette, the baseline will go near the zero. But then, the baseline will gradually go down, even overload, and there is no change in pipette resistance. If the pipette offset, the baseline will go to zero. And then go down.
"I put recording electrode into water..." How can you record pipette resistance when the conductivity of the pure water is almost infinite? In addition, there will be very large potential due to concentration gradient between pipette and the bath that will be changing with time due to continuous ion leak from pipette.
Try cleaning your Ag/AgCl electrode before putting it in the ICS. Any ICS left on the electrode will evaporate, leaving salt crystals. Next check the Ag/AgCl electrode is properly chlorided. Confirm it's not a problem with your amp by placing the Ag/AgCl directly into the bath.
Have you checked the osmolarity of your ICS? Are you using K2SO4 in your ICS? It can sometimes play up and it may be worth moving over to a K-glutamate or just KCl based ICS.
Good luck!
May be I am wrong but try to change water to extracellular solution. Use moderate possitive pressure applied to the pipette. I think that you dramaticaly change junction potential during the experiment because of local change of the solution near the pipette during the experiment (and because you use water outside). Since the pipette resistance is stable everything should be fine. Another reasons could be as mentioned - changing of chloride concentration in the solution (if you use AgCl as a reference electrode it should be sensitive), also important that you have well-chlorided electrode in the pipette.
I might not got the point right but just placing the recording electrode in water only? This doesn't give you the pipet resistance. As mentioned before you need some electrolytes in your solution and of course the reference electrode in the same chamber to close the electric circuit. Otherwise you have an extremely high resistance which cannot be compensated by the amplifier when you adjust the baseline.
If you want to measure your pipette resistance, you need to have an electrolyte solution both in the bath and in the pipette. Otherwise, the elctric circuit is open (infinite resistance) and you cannot measure anything. Your reference electrode has also to be in the bath except if you use an agar bridge.
One has to know the composition of your pipette snd bath electrolytes to answer your question.
As guess, the problem could be junction potential. I would recommend a high KCl conncentration in the reference electrode
I would personally go further and put the reference electrode in a separate chamber with the ICS, the pipette into another separate chamber containing the extracellular solution you are using, and then an Agar bridge between the chambers. This is because of junction potentials, but for comparison look at what happens if you have both electrodes in the ECS.
As mentioned above, most likely the drift you are seing comes from the different ionic composition bathing your electrodes. Thus placing ICS at both electrodes will solve the problem, (agar bridge included).
As others pointed out, you should put your electrode in a experimental solution not just in water. I think the continuous drift you saw is due to pH imbalance between the inside and outside of the electrode.
Thanks very much for all the response.
Sorry for the misunderstanding. The "water" in the question, I means normal aCSF.
Yes, I changed Ag/AgCl electrode regularly.
My ICS is using CsMeSO4.
I think there is something wrong with my ICS. aCSF and ICS cannot get to the balance. So the baseline is not stable. The liquid junction potential is offset. At first the number of pipette offset is -10mv. Then next time, it will go to -3mv and then go up to +10mv. So this means LJP is changing. Maybe Osmolarity is important. But how?
Best regards,
Mao
maybe dilution facor you must condidered for the change. The electric mesurament need to change for the change in the solution, for to the electrodo to the bath. Try usign equal solution for get zero and observed the stability
Mau, what is in your intracellular solution? Nicola is absolutely correct, if your ground or reference has dirt or lacks chloride things may drift. The same is true of your electrode wire. It must be have a thin coating or chloride on it as well. Another possibility is your intracellular solution components. Do you have chloride in your intracellular (approx. 10 mM)? If not, chloride will not be able to be exchanged in both directions between the intracellular solution and the electrode wire. This will prevent you from balancing the offset.
"I put recording electrode into water..." How can you record pipette resistance when the conductivity of the pure water is almost infinite? In addition, there will be very large potential due to concentration gradient between pipette and the bath that will be changing with time due to continuous ion leak from pipette.
You probably meant that you put your pipette into a bath solution, not pure water? if that is the case you should then measure your piptte resistance when you dip it in your bath soultion. Now, if you do still see a drift of your potential then check what Nicola said, i.e., check your reference electrode if it's well chlorided or not. If you want to know the three ways of chloriding your reference electrode, follow up to this link: https://www.researchgate.net/profile/Fouad_Lemtiri-Chlieh/contributions?ev=prf_act and download the reference Lemtiri-Chlieh and Ali (2013) with the title: Characterization of heterologously expressed transporter genes by patch- and voltage-clamp methods: application to cyclic nucleotide-dependent responses. You will find a detailed description of how to Chloride your electrode.
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