G418 mammalian cell selection question - (Jul/15/2013 )

I am using G418 400-600ug/mL to select for my transfected cells. I have used fibroblasts, epithelial and endothelial cells. I had a general question - I am seeing both my transfected (GFP tagged) cells as well as my untransfected cells dying as well as surviving from the selection treatment. I have been selecting for 14 days now.

My question is how are my uninfected mammalian cells able to survive the G418 treatment if mammalian cell do not normally carry the neo-resistant gene? My plasmids that I transfected with do contain this gene.

Hi,
I have also started working with G418 selection in adherent as well as suspension cells. Are the cells cross contaminated. Have you ever checked the GFP positivity in untransfected cells after this problem. Prepare fresh G418 and check if it works.

watsonkatherine on Mon Jul 15 22:48:03 2013 said:

You are not using a high enough concentration of G418, if your untreated cells are surviving. You need to titrate your G418 stock and find the concentration required to kill your cell line. Then use this concentration for selection of your transfected cells.

Different cell types have different LD50 and that is why for a particular cell line, you have to do a killing curve.