Re: Viability

Stuart,

in my opinion, PI (propidium iodide) is still the King of viability  
dyes...  7AAD is pretty good.  We use PI whenever possible.

However, these dyes don't work when you fix & permeabilize cells --  
they leak out too quickly.  Thus, the fixable viability dyes (which  
are covalent reactions) need to be used... this includes EMA (the  
original), and the newer amine-reactive dyes from Molecular probes.

With regard to simple fixation (paraformaldehyde) -- we found as you  
did that PI is compatible with PF fixation ... for a limited time.  If	
you analyze your cells within 2-4 hours, there is no significant  
problem.  But if you wait longer than that, the PI slowly leaks out of	
the dead cells (and into the previously live cells) rendering the  
separation far less believable.

mr

On Dec 12, 2007, at 7:17 PM, Stuart Berzins wrote:

> A couple of (perhaps) naïve questions:
>
> 7aad seems to have been around for ages and appears to give good  
> separation
> of cells that are (apparently) live and dead. Yet, there seems a  
> clear trend
> away from an old fashioned dye like 7aad to other products (eg the  
> range of
> Mol Probes viability/vitality dyes). Is there something I'm missing  
> about
> 7aad not doing the job? If the others are better than 7aad, can  
> someone tell
> me why. Is it just the colours they are detected in, or something  
> more than
> that? I want to use the best approach and happy to change from 7aad  
> if need
> be, but don't want to change for change sake.
> Any advice?
>
> On a similar topic, we stain human PBMCs with 7aad in the antibody  
> staining
> cocktail, then wash, then fix then run the samples. This gives us good
> separation of positive and negative peaks, which we take to indicate	
> cells
> that were alive versus dead prior to fixation. Are we wrong to do  
> this? I
> see lots of instances where people say things like 7aad or PI can't  
> be used
> for fixed cells. Our approach seems too obvious for people to have  
> missed,
> hence I'm wondering if there's something wrong with doing it that way.
> Anyone have any comments?
>
> Regards,
>
> Stuart
>
>
> Dr Stuart Berzins
> NHMRC RD Wright Fellow
> Department of Microbiology and Immunology,
> The University of Melbourne,
> Parkville 3010,
> AUSTRALIA.
> email: berzins@unimelb.edu.au
> Ph (office): +61-3-8344-5706
> Ph (lab): +61-3-8344-5704
> Fax: +61-3-9347-1540
> Mobile: 0427 849 123
>
>
>
>
>