AAD positive means late apoptosis? - About cells that are simultaneously positive for annexin V and AAD (May/16/2011 )

We have a discussion in the lab about apoptosis detection by annexin V/AAD labeling. Many people say that Annexin V+/AAD+ cells must not be considered apoptotic but necrotic. I agree, provided that the apoptotic stimulus was rather short (for example 2 hours-staurosporine). However, when the stimulus lasts for 24 hours (serum starvation for instance) I think that cells which started the apoptotic process shortly after the start, had enough time to cross the early apoptosis line towards the "late apoptosis" condition. And therefore they should be considered when calculating the percentage of apoptotic cells. Any comments and experiences about the topic are welcome.

7AAD+/AnnexinV+ means late apoptotic. Necrotic cells are 7AAD+/AnnexinV-.

P.S. Cell death by apoptosis is a rapid process, usually takes 15-30 minutes and 2hours incubation with such a strong cell-cycle arresting agent as staurosporin results a very "presentation-like" dot-plot of apoptotic cell distribution (for lymphocytes,for example)

If you are still not sure you should analyse your cells by caspase staining and by DNA content (i.e. PI or 7-AAD staining on fixed/permeabilised cells) to catch a hypodiploid population ("gold standart" for apoptosis)

Good luck!

Useful advices,

thanks!