Re: Viability

Hi Stuart:
The main reason people use alternatives to 7-AAD is because the 7-AAD  
staining of dead cells is not a very strong interaction and can leach  
out of the dead cells when the concentration of the dye is lower  
outside the cells then inside, such as when you wash it away for your  
fixation. The problem then becomes that the dye that has leaked out  
of the dead cells is now accessible to the fixed and hence somewhat  
permeable live cells. Traditionally people have gotten around this by  
using an equal or  higher concentration of Actinomycin D, a non- 
fluorescent version of 7-AAD, in the sample buffer to prevent this  
leaching of dye out of	dead cells. With the advent of these new  
fixable dyes which are amine reactive and a more permanent  
interaction it has become a more popular approach. They come in a  
variety of colors and provide reasonably good discrimination between  
live and dead cells. There are a few other caveats regarding 7-AAD  
that you should be aware of; the concentration of 7-AAD is important  
when doing viability as it can be somewhat cell permeant at higher  
concentrations and temperatures. The recommended amount is 1ug/ml.  
Also even though 7-AAD does excite with a 488nm laser it is not its  
optimal excitation so the emission tends to be relatively "dim".
There is nothing wrong with using 7-AAD, however, in your case I  
would verify that you are not overestimating dead cells by comparing  
your samples pre and post fixation.
Best Regards-

Joanne Lannigan, M.S.
Director, Flow Cytometry Core
University of Virginia
Jordan Hall Room 7065
1300 Jefferson Park Avenue
Charlottesville, VA 22908-0734
flowcytometry@virginia.edu
(434) 924-0274 Office
(434) 982-1071 Fax


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
>
>
>
>
>