Thank you Tom for your comments on the limitations of the DNAse technique. The SBIP methodology is superior for simultaneous multi-parameter measurements of cell proliferation and other things(cyclins, antibodies, cytokines, GFP). Strand Breaks Induced by Photolysis. No DNAse, No Acid, No Heat steps and good DNA CV's as well. Developed by the Darzynkiewicz lab. Kits produced by Phoenix Flow. The product is called Absolute-S. ______ ______ _______ /_____/ /_____ /______ C. Kevin Becker / / ______/ Phoenix Flow Systems, Inc. 11575 Sorrento Vlly. Rd. #208, San Diego, CA 92121 USA (619) 453-5095 FAX (619) 259-5268 ckb@phnxflow.com -----Original Message----- From: Tom Frey <Tom_Frey@BDIS.Com> To: cyto-inbox Date: Tuesday, February 16, 1999 11:34 AM Subject: Re[2]: GFP/BrDU/Cell cycle >Holly > >Just a warning that TO-PRO-3 is also affected by the presence of BrdU - >Cytometry 17:310-318. 7-AAD is not affected. > >Also note that whle the DNAse technique is good for preserving other antigens >(or GFP) , it can give you variable DNA content measurements for different cells >types that digest differently, and all DNA signals are often grossly decreased. > >If you have been doing the Hoechst staining on fixed cells, you can probably do >Hoechst vs PI (or 7-AAD) to look for BrdU incorporation ... although this may >have sensitivity problems with brief pulses of BrdU. Some with more experience >in that area may have some input on the minimum pulse you can see. If you have >been using H33342 on live cells you will have to figure out how to get the >second DNA dye in with decent CV. > >Tom >___________________________________________________________________________ ____ >Subject: Re: GFP/BrDU/Cell cycle >From: Derek Davies <daviesd2@icrf.icnet.uk> at INTERNET >Date: 2/15/99 8:41 AM > > > >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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