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Transfection


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#1 Mel01

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Posted 25 October 2010 - 02:50 PM

Hi,
I am trying to get a stable transfection for HPV7 cells and selected with Hygromycin. However, when I tried to transfer the cells to a bigger cell culture dish, all the cells death. I have already tried many different ways but none of them work.
1. Trypsin the cells, and then culture it with regular medium. (death)
2. Trypsin the cells, centrifuge and remove the trypsin, and then culture it with regular medium. (death)
3. Dilute the trypsin with PBS and make it to 0.05% EDTA, trypsin the cells, and then culture it with regular medium. (death)
What else can I do? I am using the Invitrogen lipofectamine 2000. The cells look perfectly fine before using the trypsin. So, I think the trypsin is the problem, and I don't know how to solve it.

Thank you.
Mel

#2 rkay447

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Posted 25 October 2010 - 03:39 PM

Could it be that what you are trying to express is toxic to the cells and killing them? Would you please elaborate on your protocol? How soon after transfection are you trying to split the cells? How soon after transfection are you adding hygromycin? What confluency are your cells at transfection? Do you have antibiotics in the media during transfection? What happens to your cells if you transfect them and don't split them? Are you sure you are seeing death rather than suspension (ie: have you stained with trypan blue)? Hmm... I'll have to think about it a bit but we may be able to help a little more if you tell us more about your protocol.




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