Apoptosis on GFP+ cells with a FACScan

Derek Davies derek.davies at cancer.org.uk
Sun Nov 13 16:20:52 EST 2005


Hi Simona,

With your setup, you could use Annexin-Cy3 for apoptotic cells (in 
the Scans FL2 channel) and 7AAD for dead cells (FL3). I would use a 
scatter gate to exclude debris/apoptotic bodies and then gate on 
GFP+ve and within those look at annexin v 7AAD. Your control will the 
the GFP+ves in a vector only sample (I am assuming these are post 
transfection rather than a stable cell line).

There are of course alternatives to looking for apoptotic changes 
other than annexin. If you suspect (or are inducing it via) an 
intrinsic pathway, you could look for changes in mitochondrial 
membrane potential using something such as TMRE.

Good luck!
Derek


>Dear flowers,
>We would like to detect apoptic cells among GFP positive cells. We 
>have only a FACScan available so no UV laser. What combination would 
>you recommend for AnnexinV/dead cells staining among all the 
>AnnexinV conjugates available through molecular probe? Should we use 
>7ADD rather than PI? What about SYTOX Green? Should we do first an 
>exclusion gate on dead cells and then plot on GFP positive cells vs 
>AnnexinV? Or the other way around gate first on GFP positive cells 
>and then plot on AnnexinV/dead cells?
>Thank you for your help,
>Simona


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Derek Davies					Voice: (44) 020 7269 3394
FACS Laboratory,			FAX: (44) 020 7269 3479
London Research Institute,		e_mail: derek.davies at cancer.org.uk
Cancer Research UK		mobile: 07790 604112
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Web Page: http://science.cancerresearchuk.org/sci/facs/

In tenebris lux
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