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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