Hi, I used HEK293 cells as an example. When I patched a cell and got a good seal (see attachment). How do I adjust series resistance compensation in order to have a successful compensation? I use Axopatch 200B (see attachment) amplifier and Clampex 10.4.
Dear Chen,
The series resistance (Rs) of the recording pipette limits the bandwidth of the whole-cell configuration making it difficult to measure rapid ionic currents such as Nav . If you are recording something of this sort then it is crucial to use compensation whenever it is possible. But in general, it is a good idea to compensate Rs, provided that it is stable. I could compensate up to 70 while recording hERG currents in HEK293 cells.
Now, how to do it and make sure it worked....I assume you are using Axopatch 200B amplifier as shown in the attached picture. Once you go to whole cells and feel the need to compensate for higher Rs
1. Locate the series ressitance comp. panel on amplifier. There are two rotatable knobs, one is for prediction another one is for compensation.. to start with set the prediction to 10% more than comppensation knob.
2. Once you set the desired compensation you shall see the change in capacitive transients.
3. Readjust the transients in the usual way (with dials in the whole cell parameter panel) you will read the new adjusted Rs value on the dial labelled as series resistance.
If the desired Ra value is reached without adding oscillations then your compensation is successful.
Hope it helps !
Best,
Nand
And also I have another questions. 1. How do I coordinate this with pipette capacitance compensation? I was told that an easy way to observe if all of these compensation is enough/correct is to observe whether the capacitance curve (shown in the attachment) becomes a straight line (or close) as much as possible. Is that correct or accurate?
2. What is the difference between compensation knob from series resistance comp panel and series resistance dial from whole-cell parameters panel?
3. What is a "desired Ra value"? I think I am confused about Rs and Ra.
oh dear read the manual of axopatch 200b . Poppers capacitance and rs compensation are different para meter. It should be compensated before whole cell
and aftr whole cell u have to again compensate the capacitance till straight line that should be accurate
Dear Cheng,
sorry for the delayed response. As Dr JN Singh suggested please consult Axon guide this shall address most of your queries.
Briefly, regarding your questions ...
pipette capacitance: It has to be adjusted when you enter in the bath solution and not to be confused with Whole cell capacitance which one has to adjust after you go whole-cell. As you mentioned, you shall see a straight line when you use series resistance and whole cell capacitance dials in a concerted manner. After you achieve the straight line you can read series resistance (MegOhm) and capacitance (pFaraday) values directly from corresponding dials. In the case of having bad access (high Ra) to the cell, you can go for additional Rs compensation to partly make up for it. When I say 'desired' Ra value then I mean a target/acceptable range you would like to be for given system/experiements, For example if I am recording INa from cultured CA1 cells, I would aim to get Ra value below 10 mOhm.
I have attached Axon guide as a link, Hope it helps!
Best luck,
Nand
http://www.psychiatry.wustl.edu/zorumski/Axon_Guide.PDF
Thanks! Your answers and Dr. Singh's help me a lot and I've read a lot from the Axopatch 200B manual. Sorry for keeping bothering you, I understand what you said above right now but one thing keeps bothering me is that I am not quite sure about the function of SEAL TEST COMMAND on the amplifier, if I switch to V-clamp mode and record the cells, I usually keep it off. Am I right or there are some other steps need to be done? I read SEAL TEST on model cell in the manual, but I mean on the real cell.
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