hek293t and geneticin G418 - (Mar/01/2007 )

Hi, I know that this has been addressed to some extent before but I am still not clear so would love some advice!!

I was wondering if anyone can tell me whether HEK293T cells are resistant to Geneticin (G418) in general. I know you can make them reisistant to Geneticin by transfection of a neomycin resistant plasmid but I want to know if they are already resistant before I start.

My other question is this: When I normally culture HEK293T cells I split them 1:3 every 2 days. I want to do a kill curve with different concentrations of Geneticin so do I stop splitting the cells and just let them overgrow (as it takes longer than 2 days to see the effect of Geneticin)? and do I change the media every 2 days with fresh media and geneticin?

Many thanks

Rich x

well the killing of the cells is better achived if the cells divide themselves. So split at higher ratio than usual.
Moreover, you're right, you have to change the medium every 2 days til the killing process seems good under the microscope

i would plate cells in a low density and change media with genticin every 3 days. You could see differences in a week.

Hi,

Do need help on this:

How do you do a killing curve? Do you count cells during each split/passage and then plot number of cells vs. a period of 2 weeks? Do I need to split at all? Do I just have estimate the % of confluency on the plate without trypsinizing the cells for counting, then plot % confluency vs. period of time?

I've read about "initial selection" at higher conc. of geneticin (e.g. 800 ug/ml) preceding the actual selection at lower geneticin (e.g. 400 ug/ml). How long is the duration of the initial selection step?

Many thanks...

Cheers

Hi rich,

This maybe a stupid question, but I just wonder why you want to check the resistance against G418 in HEK293T cells? The reason why I ask is that I am assuming that you want to make stable cell line in 293T (if not, then just ignore me, please). And it is not recommended to make stable cell line with 293T cells, isn't it? Due to the T antigen there.