During perfprmance of electrophysilogy on nuronal primery culture cell that has attached to the coveslip i could not patch it well and seal the patch also to hold for long time to measure the current of the cell is there any way to remove the cell form coverslip without any damage of cell to culture media?
Dear Nazmul,
Once u got an established neuronal culture, you can trypsinize it with 0.25% trypsin-EDTA. I have learn from my personal experience that this treatment dont damage the neurons and if you are comfortable than you can reculture the cells in culture plate wells. You can also use cell scraper if you have obtained a single monolayer sheet and dont want to trypsinize cells..
Usually we make cell culture directly on slides of circular shape that fit the chaber. So we just need to take one of this and put in the recording chamber. But I am not sure it can be useful for your situation too. In this case no treatment is needed. We just wash circular slide and put it in chamber and start recording.
Culturing the cell directly on circular glass slides is the easiest way. You don't need a specific chamber, just put the slide in the chamber and capillary forces will hold it on the bottom. You can coat your slide with whatever you were using your normal culture.
if you try to remove the cultures from the cover slips you may find it extremely difficult to patch them. Best way is to culture on the cover slips and try to find a suitable holder for this. Although, you can Trypsinize them and scrap from the coverslips.
Let me see if I understand the root problem of your question: you have neurons cultured on coverslips and you are trying to patch for a long time but the seal keeps breaking. The solution you are thinking about is to bring them off the coverslip so that you can patch them in the solution thereby removing them from the fixed surface.
Patching free cells can indeed last for a long time and by dissociating from the cover slip you remove the cell processes leading, at least in theory, to a better space-clamped cell (no processes). But in so doing you have introduced a lot of factors that makes life even worse. First, you have to subject the cells to enzymatic digestion. This likely alter (or destroy) the very proteins you are interested in exploring (I would be very critical of this in review). By the way, I do NOT recommend trypsin for this - it is far too harsh and will strip proteins off the surface indiscriminately. [When people trypsinize to prepare cells for culture the cells are given a chance to not only adhere but also re-insert their surface proteins.] If you must do this the best enzyme I have come accross is neutral dispase (I would probably get it from Worthington Enzymes - a reliable source).
But I think you are avoiding the real problem - and that is that you can not maintain a patch in situ! Most likely you have microscope, manipulator or even electrode-holder drift. Each of these has a different solution I can't discuss them all there. However, the first thing to do is to position the pipette tip in the field of view with a graticule in the eyepiece and simply determine the rate and direction of drift. After that you have to track down the source, Good luck!
Thanks all of you for your kind sugession and specially thanks to Elise Stanley for explanation the whole matter in details.........
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