Monitoring the series resistance

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patchclamper
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Monitoring the series resistance

Hi guys,

For whole cell voltage clamp experiments,  monitoring the series resistance is important for getting an idea on how well the voltage clamp is.

I monitor the Rs for the whole experiment and discard the recordings in which the Rs increased more than 20%.

Here I have several questions confused me for a long time, i will be really grateful if you guys can help me on those.

1, Base on my understanding, the aquisition softwares (eg. axograph x) calculate Rs by fitting the test pulse curve under whole cell mode and measure the fast and slow component of capancitance transient to figure out how much is Rs. I want to ask, before monitoring the Rs, do I need to compensate the pippette transient? (I compensate the pippette transient after getting a seal, then break in.)

2, When you guys monitoring the Rs by a hyperpolarizing voltage step (eg. -5mV  for 50ms), what is the filter frequency setting on your amplifier? I noticed that if I change the low pass filter from 1kHz to 5kHz, the compacitance transient of my cell will be sharper and the Rs monitored will decrease significantly. And I think use a higher filter setting should be better to obtain a true compancitance transient of the cell.

3, In what way do you prevent high Rs and keep Rs stable for a relatively long time? The tricks I can think of are to pull a bigger tip, use intracellular solution with right osmarity, prevent drifting of electrode, re-break-in when resealing happens. Any other good ways?

Thank you all.