Re: Dead Cell Marker on 633 nm
Howard Shapiro (hms@shapirolab.com)
Mon, 11 Nov 1996 10:20:11 -0500 (EST)
>I'll say.....esp. in view of what follows. How much DNA stain do
>people want to whack about, on the reasonable asumption that that
>which binds to DNA may not be frightfully good for it?
>
DNA stains used for "live-dead" discrimination are ideally supposed to stain
only "dead" cells, and either not get into "live" ones or get in in very low
concentrations. With animal cells, the entry of stains such as TO-PRO-3 and
propidium provides good evidence that there is at least one fair-sized hole
in the cytoplasmic membrane, and, although there are circumstances under
which such holes can be made to appear transiently and reclose (e.g.,
lysolecithin treatment), nuclear staining by the dyes mentioned in cells
which haven't been subjected to such conditions is generally a good
indicator of cell death. The "live" cells don't take up appreciable amounts
of dye, and, in instances where sorting of viable cells is done with DNA
stains used as "live-dead" indicators, the viability of sorted cells
generally does not seem to be compromised.
>Out of interest, what is here the 'gold standard'? We in Aber are
>beginning to have suspicions that supposedly pure(ish) TO-PRO[3],
>or likely a metabolite / degradation product thereof, penetrates (and
>ergo stains) ""live"" [i.e. not all that knackered] cells (bugs).
>
The permeability characteristics of bacteria definitely vary over a greater
range than do those of the animal (mostly mammalian) cells commonly studies
by flow cytometry. A lot of people have used ethidium as a live-dead stain
for mammalian cells, but it does get into live cells, especially at alkaline
pH, whereas propidium is much less likely to do so and should therefore be
preferred. Ethidium gets into many live bacteria. Propidium and, more
recently, the "impermeant" cyanine nucleic acid stains, e.g., YOYO- and
TOTO-1 and 3, TO-PRO-3, etc., have been considered 'gold standards' for
animal cells, but should not necessarily be thought of in the same way for
prokaryotes, plants, and perhaps some animal cell types.
-Howard
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