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Screen your stable cell lines by PCR to confirm if the gfp gene is still there.

Sarang Kulkarni

Sent from my iPhone

On 2013-06-28, at 3:02 AM, "Aryeh Weiss" <[log in to unmask]> wrote:

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> 
> This is a not-really-confocal question, but I think it is well within the list's expertise.
> 
> We have been trying to create  a stable line of B16 melanoma cells which will express photoactivatable GFP, and can be lit up whenever we like.
> So we transfected with what we think is a suitable plasmid
> (11910 pPAGFP-C1 from Addgene), and sure enough, we have  cells that survive the selection marker (Neo) while the controls die.
> So we think we have our stable cell line.
> 
> Problem is -- none of them light up. They dont light up in a widefield scope,  and they dont light up in the confocal, when zapped with 405nm
> excitation (we tried different exposures, from 100ms to many seconds).
> 
> So I must be missing something obvious, because this is not a new technology. If someone can point me in the right direction, that would be wonderful.
> 
> --aryeh
> -- 
> Aryeh Weiss
> Faculty of Engineering
> Bar Ilan University
> Ramat Gan 52900 Israel
> 
> Ph:  972-3-5317638
> FAX: 972-3-7384051