I want to stably transfect HUT78 cells (a CD4+ T cell line) with a plasmid carrying a neomycin resistance gene and will begin selection with G418 (protocol recommends 400ug/ml).
For those with experience working with this cell line:
- What G418 concentration have you found to be effective for selection in HUT78 cells?
- Would you recommend starting with a lower dose and then gradually increasing? Please give details of the starting dose and the final dose.
- How long does it typically take for untransfected cells to die off in your experience?
Any tips on optimizing the selection window or avoiding excessive toxicity would be appreciated.
Thanks in advance!
I think the best way is to make a kill curve for your cell line. You may find a concentration that worked for other researchers but may not be optimum in your case. Using minimum concentration that effectively kill non-transfected cell is recommended.
Seed cells in 12 well plate, incubate overnight, next day replace with G-418 containing media ranging from 100-1000ug/ml. Replace media every 2/3 days. Wait for 7-10 days to see the effect. Choose the right concentration for selection. The concentration that kill cells with less than 7 days should be avoided.
Alamgir Hossain Thank you. As a negative control, I suppose I can just have untransfected cells exposed to the same dosage of G418 and measure viability?
Sidney T. Sithole yes. During selection you can use non transfected cell too. This will help to find out if anything goes wrong.
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