Joe and others,
As far as the binding of Cy5 to NOD B cells goes, I can tell you the new
formulations of PE-Cy5 DO NOT correct this problem. But we already knew this
binding in the NOD was not Fc receptor mediated, so if the companies reagent
strategy was to limit Fc receptor binding, this does not address the problem
in the NOD mouse (of course, without good CD64 blocking reagents, we haven't
ruled this out, but blocking with mouse serum has no detectable affect on
this phenomenon). We have done tests with the latest generation of PE-Cy5
reagents, and still get 3 logs of signal from non-specific binding to NOD B
cells.
My guess would be that whatever the molecule on NOD B cells that binds Cy5,
it is not limited to this strain and these cells. If we could identify the
molecule, we could probably limit the deleterious effects of non-specific
Cy5 binding in many species, as it likely shares homology with similar
molecules in other mouse strains, as well as humans. Unfortunately, none of
our friendly neighborhood reagent companies see it that way, so they have
been unwilling to support investigations into identification of the
offending molecule(s). Guess they'd rather license it after it's discovered
than play a role in its actual discovery.
Kb
--
Science is built with facts as a house is with stones--but a collection of
facts is no more a science than a heap of stones is a house. -Jules Henry
Poincare (1854-1912)
Keith Bahjat
Graduate Assistant
University of Florida
College of Medicine
Gainesville, Florida
Phone: (352) 392-4887
Fax: (352) 392-5393
kbahjat@ufl.edu
on 4/19/01 7:44 PM, Joseph Webster at J.Webster@centenary.usyd.edu.AU wrote:
>
> I think we may be getting several separate issues merged together....
> They might be different manifestations of the same underlying cause,
> but I suspect not.
>
> There is the general high background that is probably Fc related.
>
> There is also a very specific effect where Cy5 binds to cells of
> certain mouse strains; I know it happens with NOD mice, and at
> least one other strain that I can't remember.
>
> The mention of CD64 binding in the Blood article is new to me, I'll
> have to follow that up and check if it is related to our problem
> with Cy5 in NOD mice.
>
> At 08:58 19/4/2001, Mark Kukuruga wrote:
>> All,
>> I don't recall specifically (hopefully, others will), but I don't
>> think it's an Fc problem but rather something about the Cy5 . . .
>
> {snippity snippity ... OUCH!}
>
> --
> Joseph Webster, Flow Cytometry Facility
> Centenary Institute, Sydney AUSTRALIA.
>
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