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We have looked at EMA staining (with UV activation) for marking
non-viable cells in samples which were subsequently fixed (as per
Reidy et al Cytometry 12, 133-9 '91). We found, as did Mark, a
background fluorescence on initially-viable cells that increased over
time roughly (where we have done a *very* approximate calibration
using Quantum beads) as follows:
FITC PE
DAY 0 <0.4 11. (All in thousands of molecules)
DAY 1 1.4 98.
DAY 7 5.5 122.
We did *not* see changes in light scatter as a result of EMA staining;
our fixation procedure reduces FSC by about 3%. Correlation between
EMA and PI for viability seemed OK.
An alternative stain we tried was 7-AAD. We intended to use the
protocol of Fetterhoff et al.(ISAC XVI '93 abstract 204B) where
Actinomycin-D is used after fixation to saturate DNA sites not already
binding 7-AAD, but found, surprisingly, that when using 7-AAD alone,
the problem of staining of the fixed but initially-viable and
therefore initially unstained cells did not arise. *No* increase in
fluorescence background of the initially-viable cells was seen over 7
days however the 7-AAD intensity dropped off by a decade or so.
\ / < Flow Systems Laboratory
Frank Battye \__/ <<<<< The Walter & Eliza Hall Institute
Robyn Muir ----------!!<<<<<<<< Voice: 61_3_345 2540
Dora Constantinou /!!\ <<<<< Fax: 61_3_347 0852
o !! \ < IN:: "facs_copy@wehi.edu.au"
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