Stable Trasfection into Hek 293and Hela cells - (Oct/14/2001 )
Hi, I would like to know the right amount of g418 (neomycin) and Hygromycin I have to use, to select stable cell line :Hek 293 and Hela.Thx.
A good explanation is summarized in Cell and Tissue Culture: Lab ProceduresEds A. Doyle, JB Grifiths and DG Newell 27D:2.4
It is advisable to perform a death curve on the cells to be transfected, for 2 major reasons (even if results for your cell line have been published):1- The active component of geneticin (powdered form of G418) constitutes about 50% of the weight with variation between batches 2- Some groups quote active concentration while others quote net weight.
To determine a suitable concentration for selection:Plate non-transfected control cells at a low density (1E4/ml for example)The next day, add G418 at 0, 100, 200, 400, 600, 800, 1000, 1200 ug/mlIncubate the cells for 6 days (may need to refeed)Check cells under microscope. The concentration of G418 which just kills all the cells is suitable for use in subsequent selection procedures.