Re: Viability

On Fri, December 14, 2007 4:23 pm, Ronald L. Rabin wrote:
> 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
>
>
> We have compared PI and 7-AAD extensively to discriminate dead/dying
cells (with permeabilized plasma membrane) of human, mouse and rat
origin. Here are our observations:

PI is superior to 7-AAD in segregating dead from live cells. Two to 3 log
difference can be easily obtained with minimal PMT voltage, It does not
spill over into other channels-FL1 (FITC) and  FL2 (TMRE) significantly.
7-AAD is very poor in segregating the dead from live cells. Even with high
PMT voltage we see only one log difference.

7-AAD is as bright as PI for confocal imaging of live cells (without
fixation). Cells can be stained and analyzed within 2 to 3 hours provided
they are kept on ice.

The advantage of 7-AAD is that it can be used in combination with PE to
determine dead cells. Cells can be stained with  PE-conjugated antibodies
and 7-AAD and analyzed without fixation on a flow cytometer. Color
compensation between PE and PI is next to impossible and you loose a lot
signals (cells) in the process whereas between PE and 7-AAD is feasible.

However, 7-AAD cannot be used in combination with TMRE to determine
mitochondrial membrane potential and dead cells simultaneously.

SJ
-- 
S. Jayaraman, Ph.D.
Associate Professor of Surgery
University of Illinois at Chicago
909 South Wolcott Avenue-704E MSB-M/C 790
Chicago, IL 60612
Phone: 312-355-5133
Fax: 312-355-1497


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