Can cells express EYFP can do the BrdU pulse?

Derek Davies derek.davies at cancer.org.uk
Wed Jan 31 04:53:59 EST 2007


Hi there,

There are a couple of possible approaches. If you are sorting the cells and
subsequently fixing in ethanol you should check whether the GFP fluorescence
actually survives the fixation - often the fluorescence is lost.
If it isn't or you are worried, there are other secondary fluorochromes you
could use to detect the BrdU depending on which cytometer you are using. For
example you could use and Alexa647 is you have a red source on your machine.
I am pretty sure that BrdU will also come conjugated to fluors other than
FITC but I haven't done that search!
Finally, you can do this without the need for sorting; fixation in a
cross-linking aldehyde fixative will preserve the GFP fluorescence (although
the signal to noise ratio may decay slightly). You can then use an enzymatic
unwinding of the DNA using DNase to get the antibody into the BrdU. The
secondary fluorochrome will depend on whether you want to combine with a DNA
binding dye but we usually use a combination of Alexa647 for the BrdU and
either PI or 7AAD for the DNA.
Hope that helps and good luck!
Derek



On 29/1/07 5:07 pm, "Junji Lin" <junjilin at gmail.com> wrote:

> hi, I recently sorted some cells expressed GFP and do the cell cycle
> analysis with it. Since most anti-BrdU primary or seconday antibodies are
> FITC conjugated.This approach seems not feasible.Does anyone have
> experiences on labeling GFP cells with BrdU and do cell cycle analysis? Or
> you know if there are any companies making suitable antibodies for this kind
> of analysis?
> Thanks!
> 
> Junji Lin
> University of Utah

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