Karim,
I assume you are trying to identify mouse DC with this staining
combination, in which case expression levels of those two markers (CD11c
and MHC II) are both quite high.
I believe the problem is the large spectral overlap between the two dyes.
As Mario and I have discussed in more detail in a review (N.Baumgarth & M.
Roederer, 2000 J. Immunol Methods 243: 77-97), the large compensation
necessary between these two dyes will broaden your negative population to
the point where it can become very difficult to distinguish positive from
negative cells. Part of the reason is the very low number of photons
measured in the far red.
I am not sure that there is a good solution to your problem other then
switching the reagents around and using with Cy7-APC a reagent that is not
expressed as highly on the cell population of interest, so that it will not
affect your APC staining quite as much.
As for the general "disappointing" staining with Cy7APC.....can you
increase laser power? All of the tandem-dyes that I have tried seem to be
doing increadibly much better at higher laser power. The more the better it
seems. This is really a limitation for use of the dyes when you try to use
them with low-power lasers.
Nicole
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>Hi,
>Has anyone ever tried to co-stain double pos. cells with one marker in
>APC, the other in APC-Cy7? We routinely costain murine MHCII and CD11c.
>Now we need those markers in APC and APCCy7 resp.
>We use a rat anti-mouse MHCII and a biotinylated hamster anti-mouse
>CD11c, followed by APC-goat anti-rat (Caltag) and streptavidin-PharRed
>(Pharmingen).
>When used separately, both colours are reasonable (although I always
>found PharRed to be quite disappointing). However when used together on
>the cells coexpressing classII and CD11c, both signals from those
>secundary reagents collapse to near oblivion...
>Could there be steric hindrance? Quenching? Or something stoopid I'm
>missing? I'd love to hear suggestions or anyone with similar
>observations!
>Karim
>
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Nicole Baumgarth
Center for Comparative Medicine
University of California
Davis, CA 95616
Tel: 530 - 754 5813 (office)
530 - 754 6538 (lab)
FAX: 530 - 752 7914
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