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Detecting GFP expression driven by mammalian promoters

'Mike' Michael J. Moser moser at U.WASHINGTON.EDU
Tue Feb 9 12:41:52 EST 1999


Malcolm,

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

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
> 
> Malcolm
> ----------------------------------------------------------------
> Dr. Malcolm S. Ogg
> Centre for Cardiovascular Genetics
> Department of Medicine
> UCLMS
> The Rayne Institute
> 5 University Street
> London
> WC1E 6JJ
> Tel: 0171 209 6977
> Fax: 0171 209 6212
> Email: rmhamso at ucl.ac.uk
> 
> 




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