Hello Holly, Interesting question! I have users who are doing BrdU and GFP simultaneously. The way I approcah this is to use the DNAse technique for unwinding the DNA to get the BrdU Ab in. This uses a paraformaldehyde/Tween fixation which preserves the GFP fluorescence nicely. I use either a PE or Cy3 secondary to visualise the BrdU. (See Carayon and Bord 1992 J Immunol Methods 147, 225-230). For short BrdU pulses where all we want is to see if there is a difference in the percentage of cells cytcling, this is fine. To date we havent tried doing a DNA stain as well. The trouble with using Hoechst here is that you would be seeing some quenching of the Hoechst by the BrdU which may complicate things. You could use 7AAD or TO-PRO-3, but I suspect that the profiles may not be the best but this is the trade off of having to use a sub-optimal fixation regime. Good luck! Derek On Thu, 11 Feb 1999, Holly Lamb wrote: > I've got an investigator who would like to look at GFP expression, BrDU > uptake and cell cycle information simutaneously. Up to this point he's > been measuring GFP with Hoechst staining for cell cycle with good success. > Now, however, the denaturation step used in the BrDU protocol has added a > considerable wrinkle to the process. We've suggested trying PI or 7AAD as > an alternative to the Hoechst for cell cycle. Can anybody suggest a good > starting point for a protocol to measure these parameters simultaneously? > Any help would be appreciated. > > Holly B. Lamb > Flow Cytometry/Optical Morphology Core Facility > 1414 Guggenheim Bldg > Mayo Foundation > 200 1st St. S.W. > Rochester, MN > 55905 **************************************************************************** * Derek Davies Voice: (44) 0171 269 3394 * * FACS Laboratory, FAX: (44) 0171 269 3100 * * Imperial Cancer Research Fund, e_mail: derek.davies@icrf.icnet.uk * * London, UK * * * * Web Page: http://www.icnet.uk/axp/facs/davies/index.html * ****************************************************************************
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