I want to do out side out patch in HEK cells having nicotinic acetylcholine receptors. Normally we get good amount of current from whole cell. we also get succeded in outside out patch but its very rare to get current. Therefore, I want to ask is there any special way to perform outside out patching. How to make cell sticked to bottom so that it doesn't come up with pipette, what should be pipette resistance or how wide pipette should be? is there any way to get out side out patch with pipette having resistance around 1 or 1.5 ?
Hello Abhilasha,
I have made o-o patches of HEK cells transfected with nAChRs for many years and have had no trouble getting substantial currents with 4-6 Mohm electrodes. It is possible that you have to alter your transfection recipe to increase the amount of receptor cDNA. Or, you may have to use some sort of co-transfected marker to identify highly-transfected cells. I have used CD8 as a marker for this (Biotechniques. 1994 Nov;17(5):876-81., PMID: 7840967). Others use GFP.
Please let me know how things work out or if I can help further.
Jim
Although I have no direct experience of recording nicotinic ACh receptor currents in outside-out patches, I would expect the channels to survive patch excision and behave normally (certainly other ligand-gated channels do).
HEKs stick well to standard tissue culture plastic dishes, you shouldn't need to coat them with any substrate (collagen or laminin or poly-L-lysine). Go whole-cell and then pull the electrode back very slowly. We do incremental pulling, meaning pull a little and wait a moment, then pull a little more and wait etc. I find that outside-outs always fail to form if you excise too quickly. You might find you are making it hard on yourself using such low resistance electrodes, but it is definitely possible to successfully excise outside-outs with 1 MOhm resistance electrodes. I would just practice first using 4-5 MOhm resistance electrodes until you have that right and then lower the electrode resistance.
Your are formulating more than one question:
1) adhesiveness of cells to the substrate,
2) single-channel recording in the inside-out configuration.
The answer to the first question depends to the type of cells. HEK cells adhere quite well to plastic substrates (e.g. Petri dishes), but if you grow cells on glass coverslips try coating them with poly-lysine or the like.
2) My best advice is to go to the sources: Try reading the paper I quote below, and then follow some of the reasonable personal suggestions of the various colleagues...
Pflügers Archiv
August 1981, Volume 391, Issue 2, pp 85-100
Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches
O. P. Hamill, A. Marty, E. Neher, B. Sakmann, F. J. Sigworth
one easy and cheap trick for cell adhesion is to wipe the coverslips with a coton cloth before autoclave. It works better than poly lisine
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