Re: Viability

Stuart,

My understanding is that DNA intercalating viability stains may miss 
some dead cells in which the DNA is too damaged or the nuclei are lost. 
  I have run into this problem as I am working with a B-cell line 
derived from a patient with Fanconi Anemia, with a mutation in FancC, 
which is involved in a DNA repair pathway.  A certain fraction of cells 
in the culture are undergoing apoptosis, likely due to irreparable DNA 
damage, and they do not stain with PI or 7-AAD.

The following reference compares the new amine reactive dyes with the 
DNA intercalating stains:

Perfetto et al. (2006). "Amine reactive dyes: an effective tool to 
discriminate live and dead cells in polychromatic flow cytometry." J 
Immunol Methods 313(1-2): 199-208.

As far as using 7-AAD with fixed cells, in the protocol in "Current 
Protocols in Immunology", they include nonflourescent actinomycin D 
during the fixation step to prevent leakage of 7-AAD from the cells.

Kendra
_________________
Kendra Hyland, Ph.D.
Discovery Genomics, Inc.
614 McKinley Place N.E.
Minneapolis, MN 55413

ph/ 612-656-4485 (office)
ph/612-379-2956 x 6566 (lab)
fax/612-379-6580
kendrah@discoverygenomics.net

http://www.discoverygenomics.net/
On Dec 12, 2007, at 6:17 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
>
>
>
>
>
>
>