Here are some answers that might not have appeared on the list - thanks
again to all who answered.
Ray
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The solution to your problem is ethidium monoazide(EMA). You incubate the
cells
pellet before permeabilisation with 10 uL of 5ugr/ml EMA under a desk lamp
for
10 minutes (minimum 5).This step will bound permanently EMA (photolabel) to
your
nucleic acids.Wash twice with 3.5ml of PBS to remove unbound EMA and proceed
to
your intracellular staining procedure.At the end of preparation fix with 1%
paraformaldehyde The dead cells before permeabilisation will show up stain
in
red in the same PMT as for ethidium bromide.(ref.C.Stewart).We try it and
were
satisfied with the results on different cells.
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Use ethidium monoazide. It enters dead cells only, but differs from PI it
that it can be cross-linked to the DNA permanently by photoactivation (under
a desk lamp). Then after washing and fixing of the cells, only those cells
that took up the ethidium monoazide before fixation will fluoresce red.
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A couple of decades ago we did some work using Calcofluor White to do
viability
analysis. It might work; you would need a UV laser, and you would need to
add
the CFW before fixing. It might be worth a try.
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I've heard of some who try using lactate dehydrogenase as a marker for
necrotic
cells as it leaks out relatively quickly when the cell dies, but I don't
know
how well it works but I think there is an antibody available.
If the labelling could be performed while the population is alive and intact
then conceivably it might be possible to use a polar reactive dye like Texas
Red
SE or AlexaFluor 594 SE. At low levels intact cells would only label on the
surface but dead cells would label more heavily as a greater number of
targets
would be accessible for the dye to attach to.
Molecular Probes offer a kit that may suit you needs in this regard. The
product is L-23102 and with luck the link below will provide more
information.
http://www.probes.com/media/pis/mp23101.pdf
One could also use an actin probe but again the live cells would have to be
intact in order to give the differential labeling.
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This question comes up regularly. The dye you want is ethidium
monoazide (EMA) and protocols for using it can be found in Current
Protocols in Cytometry and many references on the Purdue site. This
dye is added before fixation to identify the dead cells and is
cross-linked to the DNA with light. It fluoresces in the same range
as PI but is much dimmer. It really works fine when cells must be
fixed and permed.
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Why don't you gate the dead cells out using FSC vs SSC plots?
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