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Detecting GFP expression driven by mammalian promoters

Eugene Kandel u09577 at uic.edu
Tue Feb 9 15:15:03 EST 1999


Hi!

We used both EGFP and EBFP in HT1080 in a microplate reader (CytoFluor
II). In my experiments both GFPs were expressed from CMV. With both
proteins I got a near linear dependence of fluorescence on the fraction
of positive cells. 
EGFP offers much higher signal than S65T and a well with ~20% positive
cells is distinct from control. It is important to use control with the
same number of cells as in the experimental wells, because auto
fluorescence makes a noticeable contribution to the reading. I also did
triplicate wells for each point and took an average of several readings
for the same well.
Although I was able to detect EGFP even on regular multi-well plates,
but the best results were obtained on "UV"-plates (I believe they are
made by Corning). "UV"-plate were the only ones on which I could detect
EBFP. These plate are not TC-treated, but cells adhered to them anyway.
Before reading I substituted medium for PBS, but I do not know how
important it is.
Hope it helps. 

Eugene.

-- 
Dr Eugene S. Kandel
The University of Illinois at Chicago 
Department of Molecular Genetics (m/c 669)
900 S. Ashland Ave, Rm 2106
Chicago IL, 60607

312-3552024 (work)
312-6668577 (home)
312-4130353 (Fax)



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