To the ongoing discussion on light scatter changes in apoptosis let me add
some of our observations:
We do notice a transient increase in cell size (observed by microscopy)
early, at the time of the first evidence of caspases activation detected by
FAM-VAD-FMK (FLICA) binding. Some of these FAM-VAD-FMK-labeled cells are
distinctly larger that the unlabelled ones. It is difficult, however, to
correlate this increase with the increase in forward scatter measured by
flow. Perhaps indeed, the Coulter volume would be a more sensitive measure
to detect this change. However, a transient (but brief) phase of the
increase in side scatter can be more reproduciby demonstrated. In fact,
these changes were described as early as in 1991 (Swat, Ignatowicz &
Kisielow, J. Immunol. Meth. 137: 79-87). Since side scatter is a reflection
of changes in refractory and reflective properties of the cell its increase
may be caused by cytoplasm and chromatin condensation as well as by nuclear
fragmentation.
Interestingly, we observed that further progression of apoptosis may go
along two pathways: (1) When transglutaminase ("tissue" TGase) is activated
the cell dramatically shrinks, becomes irregular in shape and nuclear
fragmentation is limited (sometimes no fragmentation is seen while the
nucleus is maximally condensed). (2) When there is no evidence of TGase
activation the cell remains spherical, still relatively large, and the
nucleus is fragmented onto many chromatin pieces (Grabarek et al.,
Cytometry, in press).
I would like add a word a caution: the changes in light scatter are not
very specific for apoptosis and therefore cannot be used per se, without an
additional more specific marker, as evidence of apoptosis. Furthermore, they
may vary depending on the cell type and inducer of apoptosis. Needless to
say, there are differences between the types of flow cytometers in their
sensitivity to detect changes in forward and side light scatter.
Zbigniew Darzynkiewicz
New York Medical College
19 Bradhurst Ave.
Hawthorne, NY 10532
tel: 914-347-2801
fax: 914-347-2804
http://www.geocities.com/z_darzynkiewicz
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