RE: Ethidium monoazide
cheryl@immune.med.utoronto.ca
Tue, 30 Jan 1996 09:25:35 EST
Hi There,
One of my users does use EMA for viability gating on fixed cells, and I can
give you a qualified "yes" that it does work, at least in combination with FITC.
We have found however, that the increase in fluorescent intensity over
background is not all that great, certainly nothing like PI in fresh
preparations, and that we do seem to get some additional background over
autofluorescence. We have just learned to live with these things... it's
better than nothing. The following reference will be helpful, and take heed
where they mention that each batch of EMA must be titred as it can vary
significantly.
M.C.Riedy, K.A.Muirhead, C.P.Jensen, and C.C.Stewart: Use of a Photolabeling
Technique to Identify Nonviable Cells in Fixed Homologous or Heterologous Cell
Populations; Cytometry12:133-139(1991)
I have also heard of a couple other possibilities that I will pass on to you,
but I have no experience with them. If you try one or both let me know how
they are!
1. Christiaan N.Levelt and Klaus Eichmann: Streptavidin-Tricolor is a Reliable
Marker for Nonviable Cells Subjected to Permeabilization or Fixation; Cytometry
15:84-86 (1994)
2. T.J.Fetterhoff, S.P.Holland, K.J.Wile: Fluorescent Detection of Non-Viable
Cells in Fixed Cell Preparations: Cytometry Supplement 6, International Society
for Analytical Cytology:1993 Abstracts (No.204B)
The second reference uses 7-AAD in combination with AD. ie. After staining with
7-AAD (which binds to the dead cells), non-fluorescent AD is added to compete
for binding sites and prevent further uptake of 7-AAD.
Good Luck, and let me know how things go.
Cheryl Smith
University of Toronto
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