G418 Selection Marker for Stable Cell Lines

[admin]

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!

[Duke]

Originally Posted by admin

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!

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.

[admin]

Hello Duke,
welcome to the forums

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

Cheers