Stable transformants - determining a dose response curve for the selectable marker (Jun/05/2002 )

When carrying out a dose response curve for Geneticin- how do you decide what cells are resistant and what are sensitive?

Usually, when you do a dose response for an antibiotic, you can see that cells die progressively if they are sensible to the antibiotic. If you work with adherent cells, dead one will be floating. So, when you change the media, you get les and less cells in your wells. If cells are unsensible, you will see a proliferation as time goes by (i normally test over 4-6 days)

hi there,
i had done stable trasnfection of SP2/0 cell line. After 48 hr i had added G418 at 200mug/ml, what is the maximum concentration i can use to select clone.
When should i change the media of cells and after how many days i shall serially dilute them?
Can anybody help me out, please?
Shilpi

Hi,
resistant cells will continue growing and dividing in the presence of antibiotic, and you will get more and more dead cells with time in sensitive cell lines- I do my curves over 14 days. If you are trying to pick a concentration to select stable clones with- I normally go for the one that gives you dramatic cell death by about day 5, and no live cells at all by day 14.
As far as Shilpi's question goes: I'm not familiar with your particular cell line, but cell lines differ in their susceptibility to geneticin, and for some of my cells I have gone up to 1.2mg/ml. Change media as needed, as you would in normal tissue culture.