Leak subtraction

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Sodium_fan's picture
Leak subtraction


I am a beginner on the whole-cell patch clamp system, and I am trying to measure some currents, such as sodium and Kir currents from cardiomyocytes.

I have 2 questiions:
1. Its about the leak subtraction method, so first of all is it an accepted method which I should always use? If yes, I understand that I need to edit the protocol to have n pulses before my signal to a holding level in which I know all the channels are not active, and I read some exapmles about doing it in HEk293 cells which includes only 1 transfected ion channel, but my question is to which holding level should my leak subtraction pulses be, taking in account that in any except for the resting potential i know that an active current should exist there.

2. My second question is about quality criteria, I am trying to imose some quality criteria for choosing the good measurements for analysis. During getting the giga-ohm seal, I check on the holding and take the cell to holding potential of about -80mV. now when I conduct the "break-in" stage sometimes the baseline of the signal drops down, does that mean the attachment is leaky? if yes what is the accepted cut-off to give-up and choose another cell. is 0.1nA is good enough, is also 0.4nA good enough?
I am really confused, so when get into the cell and moves to I=0 to check the cell's resting potetial I found out that it's resting potential is -70mV so I change my holding potential to -70 and obviously it decreases the current towards less negative current. so I have another question about this, If the seal is a little bit leaky (which I don't know until know how decide about it) does this affects the measuremnt of the resting potential of the cell?

Thank you very much in advance,