Detecting GFP expression driven by mammalian promoters
'Mike' Michael J. Moser
moser at U.WASHINGTON.EDU
Tue Feb 9 12:41:52 EST 1999
I have tried to use a microplate reader to detect GFP fluorescence. I
used an HT1080 (fibroblastoid) line stably expressing S65T GFP. In this
experiment ~100% of cells are expressing GFP from the CMV promoter. I
found that my lower limit of detection was 5000 cells. Unfortunately this
is almost at confluency for this line in a 96 well plate. There were two
caveats to this experiment: 1) I wasn't using fancy trays designed for
fluorescence work and 2) I was using a FITC filter set which isn't exactly
optimum for this application. But in general my conclusion was that it
would be extremely difficult to use GFP in a plate reader based assay.
Anybody else out here have any suggestions?
Mike Moser, PhD. Tel: 206-543-6585
UW Department of Pathology FAX: 206-543-3967
Box 357705 moser at u.washington.edu
Seattle, WA 98195 http://weber.u.washington.edu/~moser
On 9 Feb 1999, Malcolm Ogg wrote:
> Dear All,
> For several months I have been trying unsuccessfully to detect the packard
> GFP variants Topaz (under the control of a mammalian promoter) and Sapphire
> (CMV promoter control vector) in several cell types using a fluorescence
> microplate reader.
> Has anyone out there had any success at all using a GFP reporter system on
> a microplate reader to examine mammalian promoters, and can anyone offer me
> any advice/help (other than give up and use luciferase/B-gal)?
> The plate reader is the Cytofluor 4000 (Perkin Elmer). The cell types
> include Hela, HepG2 and NIH-3T3s (all adherent cells).
> Can anyone tell me the best culture plates to use for assaying GFP
> expression in adherent cells (what materials do not autofluoresce across
> the GFP wavelengths)?
> Also can anyone tell me if standard any cell culture media components
> fluoresce at around the GFP wavelengths?
> Although I appreciate that other systems such as B-gal and luciferase offer
> far greater sensitivity due to enzymatic amplification, the GFP approach
> would be extremely useful for looking at the timescale of induction of the
> promoter in question by a variety of stimuli. Since luciferase and B-gal
> require cell lysis for assay a time-course experiment requires several
> batches of cells, whereas GFP would allow the examination of the response
> in the same cells.
> Cheers in advance
> Dr. Malcolm S. Ogg
> Centre for Cardiovascular Genetics
> Department of Medicine
> The Rayne Institute
> 5 University Street
> WC1E 6JJ
> Tel: 0171 209 6977
> Fax: 0171 209 6212
> Email: rmhamso at ucl.ac.uk
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