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I am an engineering student studying how electric fields affect cells, specifically the phenomena of electroporation in living cells.

I know that electroporation is widely used for introducing genes into cells, but running a full transfection experiment is not so feasible in the lab I am working in.

My question is:

If I want to demonstrate the principle of electroporation, i.e. that cells open up and intake substances from the environment, by using some kind of a dye which i can later visualize, what could be a good model for this type of experiment? Specifically - what dye, what cells could be used for this?

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Are we talking about mammalian cells or bacteria? –  bobthejoe Mar 21 '13 at 0:49
    
That's probably part of his question ;) –  Armatus Mar 27 '13 at 23:47
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1 Answer 1

You will get a lot of false-positives using the following method, and a real transfection of a fluorescent protein is always the way to go, because then you will prove that the transfection was really successful.

That said, you could try to use DAPI, followed by fluorescence microscopy or flow cytometry. DAPI is a very bright stain for DNA and cannot pass an intact cell membrane. If the cell membrane is disrupted, e. g. by necrosis, DAPI will enter the cell. I have never seen anyone trying to prove electroporation this way, but it will be worth a try. It could be that the time is too short for DAPI to enter the cell, so you'd need to tweak your setup.

I'd try the following: Prepare a cell solution with DAPI (something like 5 µM concentration should do). Split the solution into two electroporation cuvettes, perform the electroporation with one cuvette, leave the second one untouched as a control. After the electroporation, analyse the samples MOMENTARILY (means: within a few minutes) using a flow cytometer (if possible).

As for the cells, you could use anything. I suggest eukaryotic cells because they are more easy to visualize than bacteria. However, you would need a laminar flow hood for aseptic handling of cells. In theory you could also use bacteria, which are more easy to handle, but I'm not experienced with immunofluorescence in bacteria.

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