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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