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
This archive was generated by hypermail 2b29 : Sun Jan 05 2003 - 19:01:06 EST