I'm currently preparing to do a transfection of my A549 cells with a plasmid containing a neoR gene. In preparation, I am trying to determine the best G418 concentration to use to select successfully transformed cells by determining the lowest concentration of G418 that can kill all cells. I have read online that 800 ug/ml is optimal for A549. However, 6 days after testing 600, 700, 800, 900, and 1000 ug/ml (changing media every 48 hours), barely any cells have died. I can tell a difference in the growth, the control cells have grown much more than the highest treatment group, but even 1000 ug/ml just seems to be inhibiting growth and only a few cells are dying.
Has anyone else had this happen? What concentrations have you found effective in your experience?
I plan on trying again with higher concentrations of G418. I am thinking perhaps the ABC transporter activities in A549 cells could be making them more resistant?
***Also, note the A549 cells I am using are not "wt," they are engineered to express a firefly luciferase gene on their cell surface and puromycin resistance (and are continuously cultured with 1 ug/ml of puro to maintain the cell line).
Any thoughts?
Hi,
G418 is selecting very slowly. In addition from ym experience the seeding density is affecting the selection. I would repeat the experiment to detmine the needed concentration and would go even higher, in my case 1,5µg/ml finally worked.
Good luck
Sven
Thank you everyone for the suggestions. I did not see any significant cell death until day 7 when 99.9% of cells were killed by 1000 ug/ml!
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