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
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I try to take one day at a time but lately several days
have attacked me at once!
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