Detecting GFP expression driven by mammalian promoters

Beat Ludin ludin at FMI.CH
Tue Feb 9 19:35:33 EST 1999


Eugene Kandel wrote:

>Before reading I substituted medium for PBS, but I do not know how
>important it is.

The other way round rather, I guess. But yes, this is important, even 
more so for a fluorescence reader than for microscopy, and particularly 
if you grow your cells in media like DMEM with a high riboflavin content 
(riboflavin has spectral properties similar to EGFP/YFP). A common medium 
with low riboflavin conc. is Ham's F12, in case you don't want to put 
your cells in PBS. BTW, serum also has slightly fluorescent components.
 
Furthermore, I've found that one can siginificantly reduce cellular 
autofluorescence by growing the cells in Trolox-supplemented ("water 
soluble Vit E") medium, but you may have to watch out for potential side 
effects.

Also, try finding plates with low autofluorescence. Try glass, if you can 
afford it.

To optimize your system, do a series of "empty" measurements (no plate) 
to see what the intrinsic floor noise of your system is. Any signal 
significantly higher than the floor noise should theoretically be 
detectable. Do the same with control cells and try to minimize the 
background signal by applying the above measures. Depending on your 
reader you may also be able to increase the intergration time (lower the 
scan speed) to further enhance the sensitivity.

BTW, Topaz is even a fair bit brighter (in my hands) than EGFP when used 
with appropriate filters.

Cheers, 
          Beat





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