live/dead fixable dead cell stain kit

bunny bunny at cotleur.com
Thu Nov 17 00:44:51 EST 2005


Tina-
I stain simultaneously surface & Live/dead viability- it works fine.
Since the dye is dissolve in DMSO I dilute it 1:50 in staining buffer 
immediately before use;  then add 50ul to cell suspension. (This way 
the DMSO/dye is completely mixed prior to adding to cells). I add my 
antibody cocktail & incubate it all together.
Note that the dye needs 30min incubation- so if your staining protocol 
is only 15min, you may want to add the live/dead dye first.
bunny


Bunny Cotleur, M.S.
Sr Research Technologist, Dept of Neurosciences
Scientific Consultant, Flow Cytometry Core
Cleveland Clinic Foundation
Lerner Research Institute, NC30
9500 Euclid Avenue
Cleveland, OH 44195
Lab: (216)444-1164
*********************************************
Caminante, no hay camino.      Se hace camino al andar.

On Nov 15, 2005, at 6:23 PM, <Tina.Powell at UCHSC.edu> wrote:

> Hello All,
>  
> I’m trying to see if anybody has used these new LIVE/DEAD Fixable Dead 
> cells stain kits from Molecular Probes.  They are available in green, 
> red, blue, and violet fluorescent dyes.  My question is in regards to 
> the staining methods.  I’d like to use these kits for a viability 
> marker, but I’d also like to stain with some other surface stains like 
> CD3, CD4, and CD8.  The tech at Molecular probes wasn’t sure as to 
> what staining should come first…the surface staining or the viability 
> stain. 
>  
> The problem is that the viability marker will stain to all proteins…so 
> if I surface stain first, the dye will bind to the protein on the 
> antibody.  If I stain for the LIVE/DEAD first, will the dye that’s now 
> attached to the cell interfere with the surface staining?  Optimally, 
> I’d like to try to stain the LIVE/DEAD and the surface markers 
> simultaneously, but I’m not sure if this is feasible.  Please let me 
> know if anyone out there has had any experience with these new kits.
>  
> Thanks,
>  
> Tina Powell
> University of CO HSC
> Pediatric Infectious Diseases.
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