how to make stable cell using MEF cell
Posted 05-29-2008 at 02:52 AM by babangida
I am making stable MEF cell by overexpression using plasmid. And I am doing selection. Many paper showed G418 400 micro gram/ml is proper concentration. But cell is not dead after treating with G418. If somebody have experience or adivise, please help me.
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Comments
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Each cell line has its own G418 threshold. You will have to do a gradient of conc of G418 to find the least highest con of G418 at which your MEFs are still viable. Its better to do a 200-2000ug/ml conc gradient on cell, and let them grow for over a week (replenish the media after day 3-4 with correct G418 conc). Usually G418 takes lot of time to kill cells, hence atleast 1 week is advisable. This process is called making a KILL CURVE on ur cell line of interest.
In my case, I wanted to express a protien stably in HEK293 cells, and did a kill curve with G418 on them (200-2000ug/ml). Cells died at 1500 and beyond, but at 1000 and lower, they were viable. hence I chose 1500ug/ml as my conc for selecting stable clones.Posted 06-13-2008 at 10:31 PM by Sauron
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thanks for this information it will be helpful. how is your stable selection coming?Posted 06-23-2008 at 03:17 PM by admin
