Martin Offterdinger wrote:
>Hi!
>I am currently facing a similar problem-visualisation of EGFP by
>inverted microscopy. Presently I am able to see my cells, but I get
>high autofluorescence of the medium. Unlike Beat I think that the auto
>fluorescence originates from the phenol-red in the medium. (I know
>that from flow cytometry were you get quite some fluorescence in the
>green range, when you use cells that had been cultivated in the
>presence of phenol red, which can be reduced by omitting pr from the
>medium) Just a thought; Ham s F12 from GiBco has lower amounts of pr.
>compared to DMEM.....
Sorry Martin, I have to disagree with that. I've started out using
completely phenol red-free DMEM (you can get that from Gibco) to reduce
background fluorescence, but it didn't help at all. After that, I've
spent some time on a fluorometer to check out all the components of our
culture media and in the case of DMEM, riboflavin caused more than 95% of
the fluorescence!
Phenol-red itself is NOT significantly fluorescent in the wavelength
range relevant to GFP.
However, I've heard, and this is really just hear-say, that phenol red is
broken down by light, forming moderately cytotoxic breakdown products.
These might very well cause an increase in cellular fluorescence (which
you would pick up in the FACS) because cells tend to form autofluorescent
byproducts when they are under stress.
Cheers,
Beat
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