Apoptosis on GFP+ cells with a FACScan

Joanne Lannigan jl7fj at virginia.edu
Fri Nov 11 19:46:58 EST 2005


Simona :
I would use Annexin V PE with 7AAD, gate on your GFP positive cells and then
look at your Annexin V vs. 7AAD. Your double positive would be mostly
necrotic cells, although late apoptotic cells may be also double positive
but with  a lower 7AAD positivity. Your single positive AV would be
apoptotic and your double negative would be your live cells.
You could easily do a comparison with your GFP negative population by doing
the same analysis one the GFP negative gate.
Remember to do single stain controls for compensation. I also like to throw
in a sample that has been induced into apoptosis as a positive control. This
will be especially important for your single stain control for AV as you
want to have a reasonable number of positive events to compensate.

Joanne Lannigan, MS
Director, Flow Cytometry Core Facility
University of Virginia
P.O. Box 800734
Charlottesville, VA 22908-0734
Office: 434-924-0274
Lab: 434-243-2695
Fax: 434-982-1071
email: joannelannigan at virginia.edu



> From: s_zompi <s_zompi at niob.knaw.nl>
> Date: Fri, 11 Nov 2005 13:53:14 +0100
> To: Cytometry Mailing List <cytometry at flowcyt.cyto.purdue.edu>
> Subject: Apoptosis on GFP+ cells with a FACScan
> 
> 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
> 
> Simona ZOMPI
> MD,PhD
> Hubrecht Laboratory
> Uppsalalaan, 8
> 3584 CT Utrecht
> The Netherlands
> Tel: +31 (0)30 212 1846
> Fax: +31 (0)30 212 1801
> 
> 
> Support Care International
> http://www.care.org
> 
> 
> 
> 




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