In article <Csoy3y.Ds at cerc.wvu.edu>, arnold at cs.wvu.edu (Greg Arnold) writes:
> Hi! If anyone has worked with these cells, your input would be
> appreciated. Specifically, I am trying to do a kill culture with these
> cells which are to be transfected with MLV-based retroviral vectors.
> The kill cultures are to check for G418(Neo) and Hygromycin resistance.
> In some of the literature I've seen, they've used G418 concentrations
> of 1.5-2.0mg/ml. My advisor seems to think that is kind of high.
> So if you have any ideas/suggestions about culturing these cells, please
> e-mail me.
> Greg Arnold
I use mouse embryonic stem cells; I have no idea if your lines behave anything
like ES cells, but I do know that our cells are sensitive to about
300-350ug/ml G418, and 150-200ug/ml Hygromycin (don't forget that at least
with G418 there is batch to batch variation in specific activity). The high
G418 concs (above 1.5mg/ml) are necessary for lymphoid cells, which don't get
too worried by G418. I'd suggest you plate out duplicate 6-well plate
cultures with a range of concs., from zero through to 500ug/ml in 100ug
increments (you can fine tune the range in a second experiment). If you need
more than 500ug/ml G418 I'd be surprised, unless they are lymphoid cell lines.
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