Re: PFA fixation and dead cells

Dear Bunny,
   How long do you leave the fixed cells with the Live/Dead stain before 
you analyse them on a cytometer?

Molecular Probes tech support said "ideally you would run them immediately 
but should be fine the next day when kept at 4C, degradation would most 
likely occur after a couple of days".  Has that been your experience?

Also, have you used these dyes with cells that are then permeabilised for 
intracellular staining?

Many thanks,
Andy

--On Monday, October 24, 2005 2:33 -0400 bunny <bunny@cotleur.com> wrote:

> Paolo-
> I've been using Molecular Probes Live/dead kits for about 2yrs now with
> great success.   Although I use #4 (runs off the UV) I've also tried #2
>	which works well off 488nm. You incubate the cells for 30min, then are
> able to fix to analyze  later.
>
> Good Luck!
> 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 Oct 21, 2005, at 8:41 AM, paolo wrote:
>
>  Dear Fellow Flowers,
>
>  I work with HIV infected cultures and I need to determine apoptotic vs
> live vs dead cells. I use an Coulter XL with a single laser (488nm) and I
> have to fix cells with 1% PFA as they are loaded with HIV. If I use PI or
> 7AAD all cells will turn dead after fixation. Can anyone suggest
> alternatives to these dyes? I already tried EMA, but perhaps there
> something else out there that does not require the extra step with a
> light source...
>
>  Thanks in advance for all your help.
> --
>
> ============================
> Aut viam inveniam aut faciam
> ============================
> Paolo Piazza, PhD
> OFFICE: 604 Parran Hall
> tel (412) 383 - 9590
> LABORATORY: 529 Parran Hall
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> Infectious Diseases and Microbiology
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> e-mail paolo@pitt.edu
>  
>  
>  



----------------------
Andrew Herman, Ph.D.
University of Bristol
Department of Cellular & Molecular Medicine
Flow Cytometry Facility
School of Medical Sciences
University Walk
Bristol BS8 1TD
UK


A.Herman@bristol.ac.uk