Stable Cell lines

Bernard P. Murray, PhD bpmurray*STUFFER* at
Tue Nov 10 20:53:15 EST 1998

In article <l03020900b26e067ab920@[]>, Saisu at
(Sailesh Surapureddi) wrote:

> My Dear Expressionists
>         I am trying to make some stable cell lines. I have tranfected my
> constructs (has amp and neo as markers) to fibroblasts (Ca-Phos method).
> After the removal of calcium precipitate, cells were split and seeded in
> low density and treated with Neomycin sultfate (500 microgram/ml).

I assume you mean G418 or geneticin rather than neomycin as otherwise you
should see no killing in eukaryotes (see a recent thread in this newsgroup).

> I am trying to develop have confluent cells but the intresting thing is
> that there are lot of
> "islets of cells" with clear demarcations (they look kinda dark in color
> when compared to the other cells in normal microscope observations.)

Was there anything else on your plasmid apart from neoR?  Are you
expressing an exogenous gene that could change the phenotype?

Are you happy that these are not simply dead cells?  Are they clumps
of cells (living or dead) left over from when you split them?

Do you ever see this in non-transfected (and non-selected) cells at
the same passage number?  You may be seeing the cells undergo
transformation - this can be triggered at late passage in 3T3 cells by
growing them in FCS instead of calf serum.

How often do you change your growth medium?  This has to be done
fairly regularly to ensure there is active G418 present.  Also,
some people recommend increasing the concentration of drug as
the cells reach confluence as they can become more resistant
at this stage.

> 2. Can anyone suggest me on how to pick up individual islets without
> disturbing the rest (I can only see them under microscope and not
> distinguiable to normal vision)???

I've only ever picked small macroscopic colonies so I can't help.

If you are really interested in the "dark" cells then try and
enrich them by growing your plates to confluence and harvesting
them when the incidence of these foci is at its highest.  If you
see more foci in the daughter dishes it would indicate a stable
change in phenotype.
     If you are simply interested in obtaining normal clones
then split them at very low density into drug containing medium
and pick the eventual macroscopic foci.
     I hope that this helps,
Bernard P. Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA

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