4 replies to this topic

[Artzamendi]

We have transfected HEK-293 cells using a vector with the protein of interest and that confers G418 resistance. We are now performing limiting dilution methods to perform clonal selection and choose the best producers. However, when we seed cells at 1 or 5 or 10 cells/well in 96 well plates, cells do not attach, do not divide and die in 2-3 days. Any idea what can be happening? Is it worth to perform cloning calculating higher numbers of cells per well?
Thanks a lot in advance.

[bob1]

Try coating the wells with collagen or poly-lysine, this will help with attachment.  You may also need to add conditioned medium to help keep the low cell numbers alive- cells secrete things which help with attachment and growth, if you have low numbers, there won't be enough of these in the medium to keep the cells alive.

[Artzamendi]

Clever advices. I assume that when you say "conditioned medium" you mean just to pick up culture medium coming from a flask full of cells to get the growth factors etc that are absent in the non-conditioned one?
Thanks a lot for the help!

[bob1]

Correct.  You should filter the medium (0.2 um to remove any contaminating cells and debris) and store it aliquotted at -80 if you need to keep some.

[Artzamendi]

Many thanks indeed again