From: ZBIGNIEW DARZYNKIEWICZ (darzynk@nymc.edu)
Date: Thu Dec 28 1995 - 11:21:28 EST
Dr. Wortis inquires whether the frequency of "sub-G1 events" can be considered to represent apoptotic cells. 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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