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Indeed, the "sub-G1 events", in addition to being individual
apoptotic cells, may represent: (1) fragments of apoptotic nuclei,
(generally there are many fragments from a single nucleus); (2)
individual apoptotic bodies (many apoptotic bodies arise from a
single cell); (3) individual chromosomes or chromosome aggregates
released from mitotic cells following cell lysis, and (4) cellular
and nuclear fragments after mechanical cell damage, e.g. after
isolation of cells from solid tumors.
The problem is particularly severe when cells are lysed rather than
fixed, and when exponential scale (log. aplifiers) is used for DNA
measurement. We have noticed that frequency of the "sub G1 events"
may be up to 10-fold higher when cells are lysed and DNA measured
using log amplifiers compared to that when the cells are fixed in
ethanol, the degraded low MW DNA extracted with phosphate/citric
acid buffer and the cellular DNA is measured using linear amplifiers.
The methods that rely on cell lysis and measuring DNA content using
log amplifiers, thus, do not provide even an approximate estimate of
apoptotic cells. Unfortunately, in many papers that are published
this approach has been used and the claims were made that "apoptotic
cells were counted".
It is also reasonable to arbitrarily exclude objects with DNA content
lower than 5% (or even 10%) of that of cells in G1 peak. While this
may consistantly underestimate the frequency of apoptosis, it
practically eliminates counting most individual apoptotic bodies as
single apoptotic cells, which is a lesser devil. Following extaction
of the degraded DNA, the individual apoptotic bodies have generally
less than 5% of DNA compared to intact G1 cells.
Some of these strategies are described in detail in the Chapter 2
Volume 41 Methods in Cell Biology, (1994), which deals with
identification of apoptotic and necrotic cells by flow cytometry.
Zbigniew Darzynkiewicz
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