Re: Viability

If you have a UV laser, hydroxystilbamidine is a UV excited dye that is 
a non-permeable DNA intercalator as is PI.  It opens up a channel in the 
visible spectrum if you are interested.  Available through molecular 
probes.  the separation between live-dead isn't as wide as PI (about 1 
1/2 decades as I remember) but it is good enough.

ron

Mario Roederer wrote:
> 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
>>
>>
>>
>>
>>
>
>
>

-- 
Please note change in office and phone number
Ronald L. Rabin, M.D.
Senior Staff Fellow
CBER, FDA
Building 29, Room 203A
29 Lincoln Drive MSC 4555
Bethesda, MD   20892-4555
Tel:	301.435-2034
FAX:		301.480.4103