Greetings Jakob,
Since you have a pool which is GFP and "target" positive, I would
guess that you have done a drug selection for transformants.
If this is the case, then you might consider sorting onto a layer of
naive (drug sensitive) Schneider S2 cells. The cells cultures are
maintained in drug-free medium for 24h following the sort. Then the medium
is replaced with a version containing the selection agent.
We have had some success here with this method in producing specific
clones of HepG2 cells.
Also, a small paper on this type of procedure was published in
BioTechniques.
Minden, J. S.
Published Date: 01/15/1996, Research Report , BioTechniques 20:122-129
Enhanced Efficiency of Cloning FACSŪ-Sorted mammalian Cells
I will send you a PDF file under separate cover.
Cheers,
Sam
Sam Witherspoon
sw11527@glaxowellcome.com
Dept. of Receptor Biochemistry Tel. 919-483-3078
Glaxo Wellcome R&D Page 919-857-7768
5 Moore Dr. Fax 919-483-0585
RTP, NC 27709
> -----Original Message-----
> From: Jakob h. Rasmussen [SMTP:jhr@me.dk]
> Sent: Monday, November 13, 2000 6:32 AM
> To: Cytometry Mailing List
> Subject: Monoclonal Schneider S2 cells
>
>
>
> I have a question concerning the usage of FACS for creation of monoclonal
> Schneider
> S2 cells and have been encouraged by Glenn Paradis at MIT to pose my
> question here.
> We have a poly clonal cell line expressing our protein of interest and
> GFP. Since the
> level of GFP should be roughly correlated to the amount of expressed
> protein, I would
> like to sort the high GFP expressers into monoclonal cultures. The problem
> is that the
> Schneider S2 cells can't grow when they are alone so sorting them into 96
> well plates
> and waiting for them to proliferate is unfortunately out of the question.
> I have only found one reference describing the production of monoclonal
> lines, using
> mitomycin C treated feeder cells, and I therefore wonder if any of you
> should have
> experience with this or know of somebody who has.
> Please respond directly to my mailing address (JHR@me.dk) since I am not a
> part of
> the mailing list.
> I am grateful for any help that I can get.
> Regards
> Jakob H. Rasmussen
> (JHR@me.dk)
>
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