Clonal cell-lines from fibros? - I want to make clonal cell-deribvatives from mouse embrionic fibroblas (May/06/2008 )

Hi there!
I want to create a transgenic cell-line from mouse embrionic fibroblasts. Transfection seems to be ok, but I have problems with creating a clonal cell line, for these cells were not viable in less then 1000 cells/well concentration on a 96-well plate, and still they didn't really seem to be dividing cells, I reached that only in 10000cells/well concentrations, so I got a mixed population after all. Do you have any recommendations, how do I have to modify the medium to keep them alive and dividing even in a low concentration?
cheers
Judit

You could try conditioning your medium before seeding the cells at low density. Cells, especially primary lines, usually require a certain range of growth factors and other associated things in the medium. These are produced by the cells all the time, but at low density seedings there aren't enough cells around to get the levels right in the medium.

To circumvent this you can take a flask of happily growing cells and change the medium on them, 24 hours later remove the medium and filter through a 0.2 micron filter to remove any debris and living cells that could contaminate your experiment, then use this medium (containing lots of growth factors etc) to plate out your low density seeds.