Flow cytometry reagent needed

[John Ladasky]

Hello,

I have just spent two fruitless hours searching the Internet and
various catalogs.  I am looking for an anti-mouse Ig to which PE-Cy5
has been conjugated.  I can't seem to find anyone who makes it.  Why
not?

Manufacturers may have reasoned that one would only choose to use
PE-Cy5 in a three-color flow cytometry experiment.  They probably
think that, if you're doing three colors, at least two of them are
probably antibody labels.  A second-step reagent would not easily be
confined to labeling your antibody of interest.

Still, anti-mouse Ig-PerCP is available, os maybe there is some other
subtle reason I have missed.  I would rather not use PerCP, as some of
the antigens that I am examining are not very abundant.

In my case, I am using YFP as my first label, and I am trying to
perform a quantitative measurement of surface antibody staining using
another color.  The changes are rather subtle, at most five-fold and
typically two-fold.  My attempts to perform that quantitative
measurement using Cy3 or PE as the antibody labels have been
disappointing.  I believe that the compensation circuitry is adding
too much noise and systematic error to my measurements.

I have had some success using Cy5 as the second color, on a flow
cytometer with two lasers.  That flow cytometer is not especially
convenient for me to use.  On the other hand, a single-laser flow
cytometer has just become available.  I would like to try to move my
assay over to the single-laser machine.

--
John J. Ladasky Jr., Ph.D.
Department of Biology
Johns Hopkins University
Baltimore MD 21218
USA
Earth