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-   -   K562 cell culture and transfection (http://www.molecularstation.com/forum/transfection-forum/84712-k562-cell-culture-transfection.html)

krespim 02-20-2012 09:59 AM

K562 cell culture and transfection
 
I'm trying to stably transfect BACs into K562 cells but there seems to be no protocol available out there.

I've tried lipofectamine 2000 (~4E5 cells in 48 well plate), followed by 1 week selection with 400ug/ml G418, and FACS sorted individual cells to 96 wells. There were some transfected cells but the numbers were even lower then expected and I am now trying to optimise the protocol a little bit as I have many BACs that I want to stably express.

So the questions are:
1) does anyone has a protocol for BAC transfection in K563 (lipid/polymer-based reagents) or for other suspension cell line?
2) How can one get rid of dead cells in the medium during selection without losing too many live cells?
3) Any other comments or suggestions?

A somewhat unrelated question pertains the cell culture conditions of these cells (I'm used to adherent cell line culture): The cells came from ATCC and I followed their instructions (instead of their media I am using RPMI from invitrogen) but yet I noticed that cell growth is very slow and there are a lot dead cells in media from time to time. If I dilute the cells to 2E5 cells/ml media they don't seem to get to full "confluence" that is 7.5E5 without observing loads of dead cells. Even if I "clean" them by centrifugation there seems to constant cell death. Does anyone have any idea of what I am doing wrong here?


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