apoptosis
ZBIGNIEW DARZYNKIEWICZ (darzynk@nymc.edu)
Thu, 04 Jan 1996 10:39:08 -0500
In my recent note I mentioned that a combination of the use of
detergents for cell preparation (which induces lysis of unfixed
cells) and of exponential scale (log. amplifiers) for DNA measurement
does not allow one to identify apoptotic cells as the events
represented by the "sub-G1" populations. I received a couple of
querries from the readers why the log scale creates a problem in such
a measurement. The reason is that lysis of a single apoptotic cell
which has the nucleus already fragmented (nuclear envelope is
degraded) generates numerous chromatin fragments; many of them have
DNA content below 1% of the G1 peak of the nonapoptotic population.
Such events are easily measured using exponential scale (in some
published papers one can see, from the presented histograms, that the
authors had measured objects having 0.1-1.0% DNA content of the G1
peak, and identified them as apoptotic cells). When linear scale is
used, however, all such events (after the signals are digitized),
accumulate in channel #1, which generally is thresholded out. Because
the events with very low DNA content <5% or even 10% than G1 peak)
are more likely to be fragments of chromatin, isolated chromosomes,
micronuclei, etc, rather than whole apoptotic cells, it is better to
arbitrarily set a low threshold for DNA content, during the analysis
and exlude them. This is easier to perform using linear scale. Light
scatter signal of the nonfixed, detergent -lysed cells,
unfortunately, is not always adequetely informative to discriminate
between apoptotic cells, micronuclei, chromatin fragments, etc.
Zbigniew Darzynkiewicz
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