transfection - selection of trransfected SH-SY5Y cells (Aug/01/2005 )
i ve been using G418 antibiotic to select pcDNA transfected SH-SY5Y cells (human neuroblastoma cell line), but its not working out. Im using the antibiotic every 24hrs after transfection of cells till 48hrs but io still find the untransfected cells happily surviving !
This is complicating the immunostaining of the cell-coated coverslips & the western blotting thereafter.
please advice an apt way out of the problem.
Thanks U So Much !
Every 24h till 48h? Does this mean you select during only two days?
I think that selection with G418 takes usually about 2 weeks. Sorry if I understood you wrong
It certainly takes over a week. I tested my antibiotic on wild type SH-SY5Y and they lasted quite a while. Check concentrations as well, as a selcetive agent in a growing culture I use about 400ug/ml
using G418 for years, i can tell you it's at least 2weeks for a basical selection a a month to get rid of untransfected...
I use it at 500µg/ml
two advices : first do in parallel a selection on a full non transfected plate / flask. You'll be quite sure that your selection is achieved when you'll get no cells on the "control" plate.
Second : try to do several dilutions of G418 in order to know wich one is the most appropriate. For one selection i was forced to use 1mg/ml !