Rachel, Have you done this in the NOD mouse?? If so, I'd be interested what concentration you used to get this effect. The binding of Cy5 to B cells in the NOD mouse is quite dissimilar from anything seen before. This is not a subtle background shift or a widening of a negative population (as seen in C57BL/6 or Balb/c mice), but a 3.5 log positive tight population. In fact, streptavidin-Cy5 alone (no biotinylated target) isolates B cells better in the NOD mouse than any antibody (and is much cheaper!). Attempts to block this with a variety of concentrations of several different anti-CD16/CD32 antibodies has clearly shown that this binding is not to either of these Fc receptors (or their epitopes are so distinct that they do not interfere). >From our data, this binding is specifically on B cells in the NOD, and 24G2 does not affect the result whatsoever. kb -- ----------------------------------------------- Keith Bahjat Graduate Assistant University of Florida College of Medicine Gainesville, Florida kbahjat@ufl.edu ---------- >From: "Rachel M. Gerstein" <Rachel.Gerstein@ummed.edu> >To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu> >Subject: Re: Tandem conjugates question >Date: Thu, May 13, 1999, 4:40 PM > > > Using "Fc-block" (Pharmingen), the monoclonal antibody 24G2, should block > a significant amount of this background. Also, make sure the reagent does > not have aggregates by spinning in a microfuge (5000xg for 5 min.). > > The choice of lasers depends on what you want to do - so what applications > do you want to support ? > > Good luck, > Rachel > > ****************** > Rachel M. Gerstein, Ph.D. > Asst. Professor > Dept. of Mol. Gen. & Micro. > University Of Massachusetts Medical School > 55 Lake Avenue North > Worcester, MA 01655 > > > Joseph Webster wrote: > >> Hi All, >> One group here can't use PE-Cy5 dyes because they bind to the B cells >> of NOD mice. >> PE alone works fine, so the problem is either Cy5 or the combination; >> >> Does anyone know yet just what the problem is? >> >> Can anyone tell us if APC-Cy7 or other tandem dyes (or Cy7) have the >> same problem? >> >> Another question: >> Our current lasers are argon (488) and a dye (~600). >> I am (wishfully) thinking of going to a 3-laser setup with argon >> (488), HeNe (635) and a spectrum laser for choice of UV or other lines. >> Any helpful comments &/or suggestions? >> >> Many thanks, Joseph. >> >> Joseph Webster, Flow Cytometry Facility, >> Centenary Institute >> Locked Bag No.6, Newtown, NSW 2042, AUSTRALIA. >> Ph: 61-2-9565-6110 Fax: 61-2-9565-6101 >> e-mail: J.Webster@centenary.usyd.edu.au > > >
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