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Detecting GFP expression driven by mammalian promoters

David L. Haviland, Ph.D. dhavilan at IMM2.IMM.UTH.TMC.EDU
Tue Feb 9 15:21:26 EST 1999


At 09:41 2/9/99 -0800, 'Mike' Michael J. Moser wrote:
>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?

Hi All (& Mike):

>Anybody else out here have any suggestions?

Not ones that are cheap.   It depends how much GFP work one is going to do
whether or not having a custom filter made will be worth it.  But one
option is to get the broadest FITC filter you can - most I've seen are
530/30 (+/-15 nm)... 530/40 if available??   As Mike said, even at its
lowest (50% transmission) at 515nm a GFP signal will get through as  S65T'
s peak is 510.  I do know that Omega optical makes (if I recall the specs
correctly) a 510/20 or 510/10 for flow cytometric work.  

I also realize that have a custom filter made for any machine can be big
$$$...

David
=============================
 David L. Haviland, Ph.D.
 Asst. Prof. Immunology 
 University of Texas - Houston, H.S.C.
 Institute of Molecular Medicine  
 2121 W. Holcombe Blvd.  
 Houston, TX  77030 
 Internet:"dhavilan at imm2.imm.uth.tmc.edu" 
 Voice: 713.500.2413  FAX: 713.500.2424
 ------------------------------------------------------  
I try to take one day at a time but lately several days
have attacked me at once!
=============================



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