Hi Mark, I don't know what background literature you already looked up, but keep in mind that for detecting this enhanced TO-PRO fluorescence the cells should not be pulse labeled with BrdU but the cells should be grown in the continuous presence of BrdU (at low, non-toxic concentrations) and sampled at different time points (typical 4, 8, 12,24 hr etc..). The only article that comes to my mind right now (there surely will be others) is Tom Frey et al., Cytometry 17:310-318, 1994. My lab has no hands-on experience with this technique, but it resembles somewhat the BrdU-Hoechst quenching technique (although than you detect quenched fluorescence rather than enhanced fluorescence) that Peter Rabinovitch' group, Manfred Kubbies and myself and Mike Ormerod have reports on. I hope this helps, Best regards, Dirk Prof. Dirk Van Bockstaele, PhD Laboratory of Hematology Head Flow Cytometry Antwerp University Hospital Wilrijkstraat 10 B-2650 Edegem Belgium phone 32 3 821 3900, fax 32 3 825 1148 > ---------- > Van: Mark Coles[SMTP:mcoles@nimr.mrc.ac.uk] > Verzonden: zaterdag 17 februari 2001 02:22 > Aan: Cytometry Mailing List > Onderwerp: BrdU and To-Pro-3 iodide > > > Dear All, > I recently obtained a flyer from Molecular Probes claiming > that To-Pro-3 iodide preferentially incorporated into BrdU labeled > DNA. Thinking that it would be a good shortcut I have tried the dye > out to no success. I have been staining and fixing mouse thymocytes > labeled with BrdU in 1% PF. And then incubating the cells in the > presence of To-pro-iodide for 20min on ice and then running the > samples on a FACScalibur using the HeNe laser. The To-Pro-3 iodide > definetly binds to the DNA, but I see no difference between cells > that have incorporated BrdU and those that have not. Any suggestions. > > Thanks Mark >
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