3 replies to this topic

[Wing]

Hi

Does anyone have experience of creating a stable cell line and could recommend a human cell line that is easy to transfect with a plasmid and to grow during cell cloning with serial dilution. I have read that Hek293 cells are easy to transfect but don't grow well as single cells in clonal selection. Does the antibiotic for selection make any difference in how well or quickly I can get my clone I have a choice of neomycin, hygromycin and zeocin. Thanks

[cellthetruth]

according to my experience all the things you wrote are correct, all of the antibiotics are good for selection. only two things: make sure you resuspend the cells very good and dilute the cells very very high to get single colonies. to be sure you can re-select your single colonies in a second dilution series. and then... just be patient, approx. 20 days.

good luck

[bob1]

Zeocin is the more problematic of the 3 you mentioned, it kills the cells, but can leave them intact or leave protein residue on the attachment surface, which means that it can be hard to tell when zeocin has killed the cells.

By the time you have grown up to usable amounts and screened the clones from single cells, it will take more than 20 days, typically around 60 would be my estimate.

As to cell lines, what do you need from the cell (any particular cell type)? If you just need something that grows fast and will produce a lot of protein, then something like A549 or HeLa may be a good option.

[aimikins]

I have used HEK293 cells (HEK 293T in particular) and had good yield from transfected plasmid.

you can grow them in suspension, in shaker flasks, with the addition of an agent called pluronic

you just add a very small amount and it helps to keep them from clumping in the flask.

I'm also with Bob - 20 days is very, very optimistic.