It seems as if Pharmingen is now being run by BD. Anti-BrdU from BD may be the worlds most expensive Ab at ~$400 for 50ug. At that price, why put up with suboptimal Abs? Instead, buy excellent and much cheaper anti-BrdU Abs from Phoenix Flow. They have good FITC and biotin, as well as unlabeled and possibly others. Their Ab is at least as good in our hands as the BD Ab using the DNAse treatment protocol and it is cheaper and titers further. Try it. At 3:10 PM -0500 2/15/01, Plett, P Artur wrote: >Hello David > >I have never tried the BrdU stain with Hoechst, but I do see a high FITC >background in mouse cells, specially when using the isotype provided in the >"BrdU FITC set". This makes sense somewhat since the isotype is a mouse >IgG1. However, the rat-isotype also gives a high background. We have >therefore started to stain non-BrdU labeled cells with the anti-BrdU as a >background control. Additionally, in some experiments using -in vivo- BrdU >labeling, the BrdU positive population was not a separate population but >"only a shift away" from the background. This generally happened when using >the antibody in the "FITC set". > >...Incidentally, I found out that the anti-BrdU antibody in the "FITC set" >is not the same as the one in the "BrdU kit". The antibody in the kit has a >higher FITC labeling and will therefore give a brighter more separate >population, which is critical when staining few or dimm in vivo labeled >cells, while the "FITC set" works fine for in vitro labeled cells. We were >recently informed however, that the "brighter" anti-BrdU antibody will not >be available separate from the kit (for those of us who make our own perm >solutions) until the "dimmer" lots have all been sold.... > >Artur Plett, PhD >Post-doctoral fellow >IU School of Medicine Div. of Hematology/Oncology >Indianapolis, IN 46202 > > >-----Original Message----- >From: David Dombkowski [mailto:dombkows@helix.mgh.harvard.edu] >Sent: Wednesday, February 14, 2001 11:21 AM >To: cyto-inbox >Subject: Hoechst and BrdU > > > > I am trying to use a pre-incubation with hoechst 33342 to stain DNA >followed by fixation and permeabilizing with the BD Pharmingen Brdu flow >kit. After treating cells with Dnase and staining for Brdu I notice a high >background in fitc channel with BrdU-fitc staining. I would like to know if >this has something to do with Hoechst staining followed by BrdU staining >and whether it would be better to use another UV DNA dye. I would >appreciate any comments on this issue. Thanks. > >David M. Dombkowski >dombkowski@helix.mgh.harvard.edu >Flow Cytometry-Pathology-CNY rm7017 >149 13th Street >Massachusetts General Hospital-East >Boston, MA 02129 >Tel. (617)726-1683 >Fax.(617)724-3164 -- Mark Shlomchik, MD, PhD Associate Professor of Laboratory Medicine and Immunobiology Yale University School of Medicine 203-688-2089 (phone) 203-688-2748 (fax)
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