RE: Live or dead cell marker

In the situation described by Amu Sinor I would suggest to use the
viability assay based on exclusion of ethidium monoazide (EMA). The red
fluorochrome EMA, similar to ethidium or propidium iodide, is a
positively charged molecule that is excluded from live and early
apoptotic cells. It stains cells which have lost the integrity of their
plasma membrane i.e. necrotic and late apoptotic, as well as
mechanically damaged cells. This dye can be photochemically crosslinked
to nucleic acids by exposure to visible light. Cell incubation with EMA,
followed by their illumination, irreversibly labels the cells which were
unable to exclude the dye during incubation. The cells thus can be
analyzed by cytometry at a later time after their labeleing. The
photolabeling of EMA can be conveniently combined with membrane
immunophenotyping. Details of the assay were described by Carl Stewart
and his colleagues:

Riedy MC, Muirhead KA, Jensen CP, Stewart CC: Use of photolabeling
technique to identify nonviable cells in fixed homologous or
heterologous populations. Cytometry 12:133-139, 1991. 

Stewart CC, Stewart SJ: Cell preparation for the identification of
leukocytes. Meth Cell Biol 41:39-60, 1994


-----Original Message-----
From: Amy Sinor [mailto:asinor@curis.com] 
Sent: Friday, November 12, 2004 12:02 PM
To: cyto-inbox
Subject: Live or dead cell marker

Hello,
I need some help distinguishing either the live or dead population of 
cells in my cultures.  Unfortunately there are a host of issues that 
make this a bit challenging.  First my cells are already tagged with 
GFP.  Secound, I need to use a fluorescent microscope to distinguish 
these cells.  Third, I must fix the cells after I label the cells (with 
either a live or dead marker) because I will not be able to analyze all 
the plates right away.	I have tried Cell tracker Red CMTPX from 
Molecular probes, but the dead cells are also labeled lightly red.  If 
you have any suggestions using this marker or any suggestions on 
labeling either the live or dead population that would be most helpful.

Thank you,

Amy Sinor
Curis Inc
617-503-6682