Do anyone know a very good way to avoid the elevation of access resistance while doing whole cell recording for more than 30 minutes? I am recording from neurons in acute slices. I am using normal patch pipettes which resistance are 5-10MOhm.
I assume that re-adjusting the pipette position till ~10 min after getting the whole-cell is a well-known tip. If there are any other tips in your mind, please let me know.
Thank you for your attention!
I made lengthy experiments in whole cell mode in isolated kindey tubules. Well, in my experience 10 MOhm is far too small pipette for whole cell mode, it can be easily clogged with cytoplasm. For me 3-4 MOhms work much better.
Besides, if your pipette has just atmospheric pressure, substance can flow into it due to capillary forces. Therefore, it may be advisable to have slightly positive pressure in the pipette (ca 10 mm water). But then there is a danger to make it too large and casue cell swelling, so this approach requires some care.
Maybe this will help.
Hi Vlatcheslav, many thanks for your helpful comments!
As you suggested, I will try lower the resistance of pipettes.
Giving slightly positive pressure also sounds a great solution. I usually keep it atmospheric pressure. I totally agree with the possibility of capillary forces sucking cellular components into the pipette tip. Maybe, the positive pressure should be balanced with this capillary force.
Thank you again for your great advice!
You are welcome! If possible, please, share the results with us all:)
Not only the pipette tip diameter is important. In my experience the "throat" of the tip was crucial (while the Rp was the same). Make sure that it is not too narrow to avoid clogging with dense cytoplasm parts. Also I often used slight positive pressure (ca. 5-10 mbar for layer 2\3 cortical pyramidal cells) and from time to time compress the pressure hose by two fingers to prevent clogging.
But be careful with positive pressure after "break through". Though round cells look funny, they are useless for further experiment. :) Good luck!
Well, of course, positive pressure should be very moderate - just to compensate the capillary forces, but not to cause cell swelling.
Regards!
Tirgan and Viatcheslav,
Since I got your advice, I am using lower resistance pipette, and the recording is quite fine.
Many thanks for your additional advice!
I recently started to do 60 minutes recording and my pipette is also getting clogged around 45-50 min. What is readjusting pipette tip position and how it helps to lower access resistance. I am patching medium spiny neurons and I tried pipette tips from 2.2 - 5 though it is quite hard to find patchable cell below 3 megaohm pipette as those cells are mostly quite small. I tried applying positive pressure but cells are swelling and sometimes access lost ( especially with 4-5 as I assume pushed membrane clog pipette). What would you suggest to keep access with medium spiny cells?
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