Gwen Draycott writes:
[Can anyone recommend a viability dye in place of propidium iodide when
using annexin V-FITC to stain for apoptosis. I am trying to measure
apoptosis in adherent breast cancer cells treated with doxorubicin. My
dilemma is: doxorubicin has an endogeous red fluorescence that overlaps
that of PI. In addition, to top it all PI can't be used on doxorubicin-treated
cells as both the drug and the PI are DNA intercalators, which means that
competition ensues between drug and dye.
Hopefully, without giving the impression of being too picky, I need a
viability dye that will:
(a) not overlap with doxorubicin's endogenous red fluorescence
(b) not employ the mechanism of DNA intercalation so it doesn't
interfere with the cell damaging action of doxorubicin]
As an alternative to dyes that are excluded by viable cells, I suggest you
consider esterified probes that require an intact membrane for cellular
retention. In my experience dyes such as indo-1 and BCECF can give
very clean separation of live and dead cells. For use with annexin V-FITC
you could consider one of the red emitting calcium probes from Molecular
Probes, such as fura red or calcium crimson. For your particular
application these would be simply used for live/dead discrimination.
David Hedley
Ontario Cancer Institute/Princess Margaret Hospital
Toronto
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