Acridine Orange mitoses detection
ZBIGNIEW DARZYNKIEWICZ (darzynk@nymc.edu)
Sat, 23 Mar 1996 17:57:32 -0500
Dr. John Boylan inquired about the mechanism responsible for
differential staining of G2 vs. M cells by acridine orange (AO)
following DNA denaturation.
AO intercalates into ds DNA and fluorescese as a monomer, having max.
excitation approx. at 280 and emission at 530 nm. AO binding to ss
DNA which leads to red fluorescence (or more accurately red
luminescence, since it is not classical fluorescence but an emission
which more resembles phosphorescence) is more complex. Namely, AO
(which is a cation) interacting with ss nucleic acids (1:1 dye per
phosphate) neutralizes the charge of the nucleic acid. The complex
has no charge and is hydrophobic, which leads to its collapse
(precipitation). Such complex, thus, has features of the solid state,
and AO behaves in such state as phosphorescent dy is similar to the
state when aqeous AO solution is frozen. This is a triplet excitation
(intersystem crossing), and its lifetime is about 4 times longer than
the lifetime of green fluorescence of AO. As a result, there is such
huge Stokes shift, which is a result of a loss of energy of the
excited electron in triplet excitation. This is not a typical
phosphorescence, however, because the complex is still in aqeous
solution and, only the shortest-life phosphorescence can be seen,
because the longer is quenched by the collisions involving solvent
and (or) oxygen ("collision quenching"). Indeed, the quantum yield of
the red emission is very low (<10%) .
It is unclear why DNA in chromatin of mitotic cells is more sensitive
to denaturation than it is in interphase cells. We speculated that
this may be a reflection of higher torsional stress on DNA helix in
loops of the condensed chromatin. There is higher degree of
phosphorylation of histones (especially histone H1) in condensed
chromatin of mitotic cells. Because DNA in chromatin is stabilized
via ionic interactions with histones, their phosphorylation is
expected to decrease the strength of these interactions and thus
increase DNA helix stability. We have discussed these mechanisms in
detail an article in Flow Cytometry and Sorting, second edition
(Melamed, Lindmo & Mendelsohn, eds, Wiley-Liss, 1990).
The mechanism responsible for the increased denaturability of DNA in
apoptotic cells, however, appears to be different that in mitotic
cells (see Hara et al., Exp. Cell Res., 223: 732-784, 1996)
Zbigniew Darzynkiewicz
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