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The relevant literature does not tackle the good questions
about mechanism, but the observation is pretty uniform. I
listed the following 4 papers, and I should have included the
'76 Latt et al. Beyond these (and one descriptive manuscript
we have submitted) I don't know that an explanation is
available. I asked for further references on 5/11 and got a
few suggestions on apoptosis in general, but no others on the
spectral shift story.
A simple interpretation rests on the well-documented increased
permeability of apoptotic cells to Hoescht and other dyes, and
suggests that if more dye gets in then some
concentration-dependent dye-dye interaction may result in a
red shift. This is consistent with the observation that the
cells with redder emission appear only as dye loading
saturates. We have seen that dye concentration is a critical
parameter controlling the shift, but suspect that there is
more to it since adding ionomycin powerfully augments dye
loading but does not in itself shift the emmision spectrum in
healthy cells. Is a reasonable alternative found in the
apoptotic condensation of the chromatin? Can the local
concentration of dye be driven high enough to generate a red
shift?
This is a pretty significant hole in our science in my mind
since the shift is easy to measure and conveniently dovetails
into lots of other "viable" (non-fixed) cell analyses. There
must be some good fluorescence/nucleotide chemists out there
who know more than me about this, let's hear from you. If
not, I think there's some good work to do.
Watson, J. V, A. Nakeff, S. H. Chambers, and P. Smith. 1985.
Flow cytometric fluorescence emission spectrum analysis of
Hoescht-33342-stained DNA in chicken thymocytes. Cytometry
6:310.
Stokke, T. and H. B. Steen. 1985. Multiple binding mondes for
Hoescht 33258 to DNA. J. Histochem. and Cytochem. 33:333.
Ellwart, J. W, and P. Dormer. 1990. Vitality measurement
using spectrum shift in Hoescht 33342 stained cells. Cytometry
11:239.
Belloc, F. P. Dumain, M.R. Boisseau, C. Jalloustre, J.
Reiffers, P. Bernard and F. Lacombe. 1994. A flow cytometric
method using hoescht 33342 and propidium iodide for
simultaneous cell cycle analysis and apoptosis determination
in unfixed cells. Cytometry. 17:59.
Latt, S. A. and G. Stetten. 1976. Spectral studies on 33258
Hoescht and related bisbenzimidazole dyes useful for
fluorescent detection of deoxyribonucleic acid synthesis. J.
Histochem. Cytochem. 24:24.
Sorry about the bandwidth, but I have to check out my new
e-signature, because
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