G418 Selection Marker for Stable Cell Lines
 

Good morning to everyone,
Im going to try and establish a stable cell line after transfection of my plasmid.

Now for G418 selection, how much G418 should I be using to select in Hela cells and NIH 3T3 cells?

Some use 400ug/ml to select cells but doesnt this depend on many factors? How to optimize this?

Thanks in advance!

Re: G418 Selection Marker for Stable Cell Lines
 

For each cell line and sometimes for even each batch of G418 you have to determine a kill curve.
Simply seed some native cells in a 12 well plate, let them attach and add different concentrations of G418 to each well (e.g. 0, 10, 50, 100, 200, 300, 500, 700, 1000 µg/ml).
For selection of your clones after transfection use the concentration in which no cell did survive the upper conditions after 3-5 days.
Don´t use to high concentrations (e.g. cell death in the kill curve after 1 or 2 days).
Later, once the cell line is etablished, you can lower the G418 concentration to a fifth of the selection concentration for cultivation.

I have experienced that the specific activity of G418 varies a lot from batch to batch. Therefore it is very well possible that a concentration once determined as good for selection can kill with the next batch of G418 the same cells. Therefore it is advisable to make a new kill curve with every new batch of G418 and not to rely on published data.

Re: G418 Selection Marker for Stable Cell Lines
 

Hello Duke,
welcome to the forums :)

Thank you for a very detailed answer. This will help me and many others!

Cheers

Re: G418 Selection Marker for Stable Cell Lines
 

thanks