No, I was not aware that the effect in NOD mice could not be blocked with anti-Fc - I stand corrected. Rachel Keith Bahjat wrote: > 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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