From: /G=Gerhard/S=Nebe-von-Caron/OU=1890CHPE/O=TMGB.URC/@LANGATE.gb.sprint.com
Date: Wed Jan 31 1996 - 06:36:00 EST
Funnily I don't recall the original posting of that message
so I don't have the sender. Could s/he please tell me
whether s/he got any error message back from our server? I
had some incompatibility for quite some time with Miller at
Procter&Gamble (perhaps no wonder regarding the
'compatibility' of our companies).
Regarding the problem of identifying cells that were viable
prior to fixation I had developed a system on bacteria that
has never been published. It can be used together with a
monoclonal antibody indirect labelled with goat anti
mouse*PE and propidium iodide for identification and total
cell detection after fixation. Cells are stained with the
first antibody and CCFAS that is:
5,6-Carboxy-diChloroFluorescein-diAcetate-Succinimidylester
(from Lambda Fluorescence Technology Graz, Cat No. LA-57)
for esterase activity / dye retention. The dichloro
derivative lowers the pK to 4.2 to avoid pH quench, the
succinimidylester creates a covalent bond inside the cell
(with time). Cells are washed twice and stained with the
goat anti mouse*PE washed two more times, fixed and measured
in the presence of 1ug/ml PI.
An example of Carboxyfluorescein, PE and PI without fixation
is published in 'Viability assessment of bacteria...' last
year in the journal of microscopy, showing that the
combination works. The compensation on leukocytes is more
tricky but possible if the stain is not to strong. We also
used the method together with fluorescence microscopy to
optimise the preparation of skin samples to find out how
many cells were dead on arrival and how many we killed in
the preparation but that also was never published.
I still look for some volunteer who would want to try the
combination in an NK-assay.
Gerhard Nebe-v.Caron
Unilever Research, Colworth Lab.
Sharnbrook, Beds. GB-MK44 1LQ
Tel.:+44(0)1234 222066
Fax.: 222344
Gerhard.Nebe-von-Caron@urcgb.sprint.com
______________________________ Reply Separator _________________________________
Subject: Re: Ethidium monoazide
Author: rolivier@pasteur.fr at INTERNET
Date: 30/01/96 20:48
>I wish to be able to identify dead cells in samples of fixed cells. The
>cells are HIV infected so they have to be fixed before running on the
>machine. I seem to remember ethidium azide can do this, has any one any
>experience of this dye? Who sells it? How do you use it?
>
>Simon Monard
>Aaron Diamond
>New York
You can effectively use Ethidium monoazide. The way to do it is to label
the cells and then to fix them.
You will find a protocol in : Cytometry 12, 133 (1991) M.C. Riedy et al.
See the Medline reference below.
I guess that you can get some more details getting in touch with Molecular
Probes, since they have this reagent in their catalog (cat.: E-1374).
Molecular Probes e-mail: tech@probes.mhs.compuserve.com
phone: 503-465-8338 / fax: 503-344-6504 or 1-800-438-0228
Good luck !
Rene Olivier
-----------------
ARTICLE TITLE: Use of a photolabeling technique to identify nonviable
cells in fixed homologous or heterologous cell populations.
ARTICLE SOURCE: Cytometry (United States), 1991, 12(2) p133-9
AUTHOR(S): Riedy MC; Muirhead KA; Jensen CP; Stewart CC
AUTHOR'S ADDRESS: Roswell Park Memorial Institute, Department of Flow
Cytometry, Buffalo, New York 14263.
MAJOR SUBJECT HEADING(S): Affinity Labels [diagnostic use]; Azides
[diagnostic use]; Cell Survival; Flow Cytometry; Fluorescent Dyes
[diagnostic use]
MINOR SUBJECT HEADING(S): Antibodies, Monoclonal [diagnostic use];
Fixatives [pharmacology]; Immunophenotyping; Lasers; Specimen Handling
INDEXING CHECK TAG(S): Human
PUBLICATION TYPE: JOURNAL ARTICLE
ABSTRACT: Flow cytometric determination of viable versus nonviable cells
in fixed samples can be accomplished by utilizing the irreversible binding
of photoactivated ethidium monoazide (EMA). EMA is a positively charged
molecule which is excluded by cells with intact membranes (viable cells),
included by cells with damaged membranes, and can be photochemically
crosslinked to nucleic acids using visible light. EMA fluorescence can be
excited using a standard argon laser operating at 488 nm and is able to be
distinguished from fluorescein and phycoerythrin. Fixation is important
when analyzing cells from a potentially infectious origin. EMA is
photochemically crosslinked and therefore unable to leak out of cells when
removed from the extracellular media, unlike propidium iodide (PI) or other
viability stains, which were heretofore commonly used. We demonstrate the
usefulness of EMA in combination with fluoresceinated and phycoerythrin
labeled monoclonal antibodies in immunophenotyping. The photoaffinity
labeling technique allows for a quick and efficient means of identifying
nonviable cells which cannot be distinguished on the basis of
light-scattering properties.
MEDLINE INDEXING DATE: 9109
ISSN: 0196-4763
LANGUAGE: English
UNIQUE NLM IDENTIFIER: 91266648
CAS REGISTRY/EC NUMBER(S):
PS: there is a problem with your e-mail address (monard@adarc.nyu.edu)=
,
you are probably using a special character that is not accepted by servers:
The original message was received at Tue, 30 Jan 1996 12:12:33 +0100
from [157.99.72.146]
----- The following addresses had delivery problems -----
<monard@adarc.nyu.edu> (unrecoverable error)
----- Transcript of session follows -----
... while talking to phri.nyu.edu.:
>>> RCPT To:<monard@adarc.nyu.edu>
<<< 550 <monard@adarc.nyu.edu>... User unknown
550 <monard@adarc.nyu.edu>... User unknown
____________________________________________________________________________=
__
Rene Y. Olivier
Unite d'Oncologie Virale
Institut Pasteur
25-28 rue du Dr Roux
75724 Paris Cedex 15
France
Tel: 033 1 45 68 83 75
Fax : 033 1 40 61 34 65
Secr: 033 1 45 68 87 41
E-mail: rolivier@pasteur.fr
____________________________________________________________________________
__
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