Zbigniew Darzynkiewicz

                                                The Cancer Research Institute, New York Medical College, Valhalla, N.Y. 10595

Address Correspondence to: Dr. Z. Darzynkiewicz
The Cancer Research Institute
New York Medical College
100 Grasslands Road
Elmsford, N.Y. 10523
tel: 914-347-2801
fax: 914-347-2804
e-mail: darzynk@nymc.edu


ABSTRACT. Two alternative modes of cell death can be distinguished, apoptosis and accidental cell death, generally defined as necrosis. Flow cytometry is the methodology of choice to study various aspects of cell death including detection and quantitation of apoptotic or necrotic cells. It offers all the advantages of rapid, multiparameter analysis of large populations of individual cells to investigate the biological processes associated with cell death. Numerous cytometric methods have been developed to identify apoptotic and necrotic cells which are widely used in various disciplines, in particular in oncology and immunology. The characteristic changes in cell size, shape and morphology as well as in plasma membrane structure and transport function, function of cell organelles, in particular mitochondria, DNA stability to denaturation and endonucleolytic DNA degradation which occur during apoptosis, all are the features used to identify apoptotic and necrotic cells by cytometry. The principles of analytical methods to detect apoptosis or apoptosis based on these changes are described and the methods are critically reviewed. Applicability of these methods both in the research laboratory and in the clinical setting is discussed. Particular attention is focused on improper use of these methods and on data interpretation. Multiparameter analysis of various molecular and biochemical features of the dying cells as offered by flow cytometry opens new possibilities in investigation of molecular mechanisms in necrobiology.


Cell Necrobiology. Mechanisms associated with cell death have recently become a center of attention of researchers in a variety of diverse fields, such as cell and molecular biology, oncology, immunology, embryology, endocrinology, hematology and neurology. Various aspects of cell death including molecular, biochemical and morphological changes which not only occur in the dying cell but also predispose the cell to respond to an environmental or intrinsic signal by death, regulate the initial steps leading to irreversible commitment to death and activate the post-mortem cell disposal machinery are the subject of intense studies (1-11). Because the scope of this field is already so extensive, involves many disciplines, and is directly correlated with a variety of life processes associated with cell cycle or differentiation, to define this field we have recently introduced the term "cell necrobiology" (5). Cell necrobiology comprises various modes of cell death, the biological changes which predispose, modulate, precede and accompany cell death, as well as the consequences and tissue response to the cell death. This term, which combines necros (death) and bios (life) may appear contradictory. The inconsistency, however, is more apparent than real. Namely, in preparation for and in early stages of cell death a complex cascade of biological processes, typical of cell life, takes place. These processes involve activation of many regulatory pathways, preservation and often modulation of transcriptional and translational activities, alteration of cell organelles activity, activation of many diverse enzyme systems, modification of the cell plasma membrane structure and transport, etc. Of particular interest are changes in proteins whose function is to regulate the cells proclivity to apoptosis such as bcl-2 (12-21) and ICE proteases (22-24) families of proteins. A term that refers to the biology of cell death, thus, is not a contradiction.

Cell death culminates with irreversible cessation of biological activity. It is often difficult to define at which point a cell has passed the point of no return in the death process and which changes cannot be reversed. As an operational definition of cell death, independent of the techniques measuring it, one may accept the passage of the cell through such a point. Beyond such point, the passive degenerative post-mortem changes take place

Apoptosis and Necrosis. Apoptosis and necrosis are two distinct, mutually exclusive, modes of cell death (reviews, 1-11). Apoptosis, frequently referred to as "programmed cell death", is an active and physiological mode of cell death, in which the cell itself designs and executes the program of its own demise and subsequent body disposal. A multistep complex mechanism regulates the cell's propensity to respond to various stimuli by apoptosis, whose complexity has recently become apparent (12). The regulation system involves the presence of at least two distinct checkpoints, one controlled by bcl- 2/bax family of proteins (13-17), another by the cysteine- (caspases) (22-24) and possibly by serine- (25-28) proteases. Through several oncogenes (e.g. c-myc) and tumor suppressor genes (e.g. p53), this system interacts with the machinery regulating cell proliferation and DNA repair. Regulatory mechanisms associated with apoptosis are the subject of recent reviews (9,12). Several review articles discuss antitumor strategies based on modulation of the cell's propensity to undergo apoptosis, a subject of great interest in oncology in recent years (6,29-33).

A cell undergoing apoptosis activates a series of molecular and biochemical events which lead to its total physical disintegration. Because many of these changes are very characteristic and appear to be unique to apoptosis, they have become markers used to identify this mode of cell death biochemically, by microscopy or cytometry. One of the early events is cell dehydration. Loss of intracellular water leads to condensation of the cytoplasm which results in a change in cell shape and size: the originally round cells may become elongated and generally, are smaller. Another change, perhaps the most characteristic feature of apoptosis, is condensation of nuclear chromatin. The condensation starts at the nuclear periphery and the condensed chromatin often takes on a concave shape resembling a half-moon, horseshoe or sickle. The condensed chromatin has an uniform, smooth appearance, with no evidence of any texture normally seen in the nucleus. DNA in condensed (pycnotic) chromatin exhibits hyperchromasia, staining strongly with fluorescent or light absorbing dyes. The nuclear envelope disintegrates, lamin proteins undergo proteolytic degradation, followed by nuclear fragmentation (karyorrhexis). Many nuclear fragments, which stain uniformly with DNA dyes and thereby resemble DNA droplets of different sizes, are scattered throughout the cytoplasm. The nuclear fragments, together with constituents of the cytoplasm (including intact organelles), are then packaged and enveloped by fragments of the plasma membrane. These structures, called "apoptotic bodies", are then shed from the dying cell. When apoptosis occurs in vivo apoptotic bodies are phagocytized by neighboring cells, including those of epithelial or fibroblast origin (i.e.not necessarily by "professional" macrophages), without triggering an inflammatory reaction in the tissue (reviews, 2,3,7,8,10,11).

Activation of endonuclease(s) preferentially cleaving DNA between the nucleosomes is another characteristic event of apoptosis (1,3,11).The products of DNA degradation are nucleosomal and oligonucleosomal DNA fragments (180 bp and multiplicity of 180 bp) which generate a characteristic "ladder" pattern during agarose gel electrophoresis. Because the DNA in apoptotic cells is partially degraded, the fraction of low molecular weight DNA can be extracted whereas the nondegraded DNA remains in the cell (34). In many cell types, however, DNA degradation does not proceed to nucleosomal sized fragments but stops in generating 300 to 50 kb size DNA fragments (35).

Another characteristic feature of apoptosis is the preservation, at least during the initial phase of cell death, of the structural integrity and most of the plasma membrane transport function. Also, cellular organelles, including mitochondria and lysosomes remain preserved during apoptosis. The mitochondrial transmembrane potential of mitochondria, however, is markedly decreased (36-39). Release of cytochrome c from mitochondria, believed to be modulated by bcl-2, appears to be one of the earliest events of apoptosis, triggering activity of caspases and other downstream apoptotic effectors (18-21). Other features of apoptosis include mobilization of intracellular ionized calcium (40), activation of transglutaminase which crosslinks cytoplasmic proteins (41), loss of the microtubules (42), loss of asymmetry of the phospholipids on plasma membrane leading to exposure of phosphatidylserine on the outer surface (43), and other plasma membrane changes which precondition remnants of the apoptotic cell to become a target for phagocytizing cells. The duration of apoptosis may vary, but generally is short, even shorter than duration of mitosis (7,10). Thus, under conditions of tissue homeostasis, when the rate of cell death is balanced by the rate of cell proliferation, the mitotic index may exceed the index of apoptosis.

While apoptosis is characterized by an active participation of the affected cell in its own demise, even to the point of triggering (in some cell systems) the de novo synthesis of the effectors of cell death, necrosis is a passive, catabolic and degenerative process. Necrosis generally represents a cell's response to gross injury and can be induced by an overdose of cytotoxic agents. If apoptosis can be compared to cell suicide, necrosis is accidental death and is often referred as "cell murder" or "accidental cell death" (8). The early event of necrosis is mitochondrial swelling followed by rupture of the plasma membrane and release of cytoplasmic constituents, which include proteolytic enzymes (8,10). Nuclear chromatin shows patchy areas of condensation and the nucleus undergoes slow dissolution (karyolysis). Necrosis triggers an inflammatory reaction in the tissue and often results in scar formation. DNA degradation is not so extensive during necrosis as in the case of apoptosis, and the products of degradation are heterogenous in size, failing to form discrete bands on electrophoretic gels.

Different Patterns of Apoptosis: Early and Delayed Apoptosis. Homo-phase, Homo-cycle and Post-mitotic Apoptosis. Many cell types, cells of hematopoietic origin in particular ("apoptosis primed cells"), undergo apoptosis rapidly, within few hours following exposure to relatively high concentration of cytotoxic agents (44-46). This is an example of early apoptosis which generally occurs during the same cell cycle, or even the same phase as when the damage was induced in the cell, i.e. prior to mitosis. For example, early apoptosis of S phase cells in HL-60 leukemic line cultures treated with 0.1- 2.0 µM DNA topoisomerase I inhibitor camptothecin, seen as early as 2 - 3 h after cells' exposure to the drug (44-46), occurs prior to their entrance into G2 phase. To define apoptosis which occurs in the same phase of the cycle in which the cells were initially exposed to the apoptosis triggering agent, we proposed the term homo-phase apoptosis (32). During homo-phase apoptosis, the cells remain arrested at a particular phase (or traverse it slowly) and die without progressing into the next phase of the cycle. Sometimes, however, it is difficult to ascertain whether the cells indeed underwent apoptosis during the same phase or moved to the next one. Therefore, the term homo- cycle apoptosis, was proposed (32) to denote apoptosis occurring in the same cell cycle in which the cells were initially exposed to the cytotoxic agent, without specifying the cell cycle phase. In other words, the cells die prior to, or during the first mitosis after induction of the damage. Interphase cell death, the term used in radiobiology to describe the death of cells, following their irradiation, which occurs prior to their division (47), if occurs by apoptosis, would be synonymous with homo-cycle apoptosis.

In contrast to homo-cycle apoptosis, the term post-mitotic apoptosis is proposed to define apoptosis occurring in the cell cycle(s) subsequent to the one in which the cells were initially exposed to the damaging agent (32). Post-mitotic apoptosis represents a delayed apoptosis which often occurs when the cells are pulse-exposed to relatively low concentration of cytotoxic agents and then allowed to grow in the drug free media. Post-mitotic apoptosis, thus, would be synonymous with another term used in radiation biology, reproductive cell death (48), if the death is by mode of apoptosis. It is likely that post-mitotic apoptosis results from the damage to genes which are essential for cell survival and is triggered by lack of the functional products of these genes in the subsequent cell cycles. Also, the originally sublethal damage may be amplified and/or become a lethal lesion during the next round of DNA replication and mitosis. In addition, severe growth imbalance may occur when the cell is arrested in the cycle but RNA and protein synthesis continue. The secondary changes in these cells, resulting from DNA damage and growth imbalance often cause an alteration in cell morphology and metabolism to such an extent that it is difficult to classify their mode of death. Such cells may show the features of both, apoptosis and necrosis, or have very atypical changes which lack the key morphological or biochemical features of apoptosis (47,49,50)

The proposed classification has certain merits. First, the subdivision may reflect the differences in mechanisms triggering apoptosis. During homo-phase apoptosis, for example when it occurs during S phase, the cells do not traverse G1 or G2 checkpoints. Therefore, p53, which controls these checkpoints (review, 51), may not play a dominant sensitizing role, as it otherwise does for the cells that traverse these checkpoints. During post-mitotic apoptosis, on the other hand, most likely the apoptotic machinery is actually triggered at the cell cycle checkpoints. Antitumor strategies involving p53 and the cell cycle checkpoints, therefore, may be different for homo-phase vs. post-mitotic apoptosis. Another difference which may be of relevance in oncology, is related to the fact that in the case of post-mitotic apoptosis, the cells do progress through the cycle after the initial exposure to the drug. It is possible, therefore, to additionally modulate their sensitivity during this transient time. A strategy may be developed to protect and rescue normal cells during chemotherapy (e.g. by arresting them with non-toxic agents to give time for DNA repair), in order to lower drug toxicity. Conversely, tumor cells which escape the post-mitotic apoptosis can additionally be treated with a drug which affects cell cycle traverse.

A pattern of cell death not always may have the classical features of either apoptosis or necrosis. Numerous examples of cell death have been described where the morphological and/or biochemical changes neither resembled typical apoptosis nor necrosis but often had features of both (50,52-58). In some cases, the integrity of the plasma membrane was preserved but DNA degradation was random, without evidence of internucleosomal cleavage. In other situations, DNA degradation was typical of apoptosis but nuclear fragmentation and other features of apoptosis were not apparent. Generally, while most hematopoietic lineage cell types are "primed" to apoptosis and their death has typical apoptotic features, the mode of death of epithelial type cells is often difficult to classify. Furthermore, certain drugs which trigger apoptosis may additionally affect molecular and morphological features of the dying cell thereby confusing the pattern by which apoptotic mode of cell death is recognized. For example, when apoptosis is triggered by drugs affecting cell structure and function, or by drugs affecting one of the pathways of the apoptotic cascade (e.g. inhibitors of proteases), particular features of apoptosis related to this pathway (e.g. proteolysis of the nuclear envelope) may not be apparent (27). Likewise, prolonged cell arrest in the cell cycle induced by certain drugs leads to growth imbalance which alters cell biochemistry and morphology (59).


The critical development which stimulated wide interest in cell necrobiology in different fields was the realization that apoptosis, whether it occurs physiologically or is a manifestation of a pathological state, is an active and regulated mode of cell death. The regulation consists of several check-points at which a variety of the interacting molecules either promote or prevent apoptosis (9,12). A possibility, therefore, exists for intervention, to interact with the regulatory machinery and thereby modulate the cell propensity to respond to the intrinsic or exogenous signals by death. Such a possibility is of obvious interest in oncology. The strategies of modulation of sensitivity of tumor and/or normal cells to antitumor agents, via the regulatory mechanism of apoptosis, to increase efficiency of the treatment and to lower toxicity to the patient are currently being explored in many laboratories (5,6,16,30-33). Identification of the gene protecting cells from apoptosis (bcl-2) as an oncogene (60) made it apparent that not only the change in rate of cell proliferation but also the loss of their ability to die on time may be a cause of cancer. It also become apparent that tumor progression and the increase in malignancy may be associated with the change in propensity of tumor cells to undergo spontaneous apoptosis (61-63). Of interest in oncology, therefore, is prognostic value of the rate of spontaneous apoptosis in tumors as well as of apoptosis induced by the treatment. In the latter case, the effects of antitumor agents in terms of induction of apoptosis can be analyzed in the course of therapy, thereby providing a possibility of rapid assessment of their efficiency (32,64-67).

Immunology is another discipline where apoptosis is of great importance. This mode of cell death plays fundamental role in clonal selection of T cells and is implicated as the key event in many other normal and pathological reactions (2,4,68). In particular, the mechanism of cell kill by NK lymphocytes is based on use of the apoptotic effectors machinery (69). Furthermore, since progression of AIDS appears to be correlated with the rate T cells apoptosis, attempts are made to monitor apoptosis of these cells as a sensitive marker of the disease progression (70,71).

The key role of the programmed cell death in tissue and organ development was recognized very early by embryologists. Apoptosis plays a role not only in normal tissue and organ modelling during embryogenesis, but likely, is also triggered by environmental toxins where it may be ectopic or unscheduled, leading to congenital malformations.

Male fertility is still another field where apoptosis appears to be of particular interest. It has been previously observed that DNA in chromatin of abnormal infertile sperm cells is, in contrast to normal sperm cells, very sensitive to heat or acid induced denaturation (72). This feature strikingly resembles DNA in chromatin of apoptotic cells (73,74). A correlation has been later observed between the increased DNA denaturability in infertile sperm cells and the presence of extensive DNA breakage, which is another feature of apoptosis (75). It was proposed, therefore, that apoptosis may be triggered to eliminate cells bearing e.g. DNA mutations even at late stages of spermatogenesis (75). However, in cells differentiated to such an extent many apoptotic effectors may already be absent. Therefore, apoptosis in these cells may be incomplete, resulting only in activation of endonuclease which causes massive DNA degradation and elimination of the cell in terms of its reproductive capacity. The reproductively inactive spermatozoa may still have mitochondrial activity, normal motility and, in some cases, even morphology (72). It should be stressed that the increased in situ DNA denaturability in sperm cells, assayed by flow cytometry, has become intensively used as a marker of infertility and in toxicology studies to assay the genetoxic effects of environmental agents (76,77).

Flow cytometry has become a method of choice for analysis of apoptosis in a variety of cell systems (reviews: 5,78,79). Methods chapters on use of flow cytometry to analyze the modes of cell death have been recently published (80-82). A variety of flow cytometric methods have been developed and new methods and modifications of the established assays are being introduced at rapid pace. The present article provides background information on apoptosis and on applicability of the cytometric methods in this field, as well as updates the earlier reviews on this subject (5,78,79).


Changes in morphology of the dying cell can be detected by analysis of a light scatter signal by flow cytometry. The cell traversing through focus of a laser beam in flow cytometer scatters the laser light. Analysis of the scattered light provides information about the cell size and structure (83). While the intensity of light scattered at a forward direction correlates with cell size, the intensity of scattered light measured at a 900 angle to the laser beam (side scatter) correlates with granularity, refractiveness and the presence of intracellular structures that can reflect the light (83). The cell's ability to scatter light is altered during cell death, reflecting the morphological changes such as cell swelling or shrinkage, breakage of plasma membrane and in the case of apoptosis, chromatin condensation, nuclear fragmentation and shedding of apoptotic bodies.

Cell necrosis is associated with an initial increase and then rapid decrease in the cell's ability to scatter light simultaneously in the forward and right angle direction (80,82). This is a reflection of an initial cell swelling followed by plasma membrane rupture and leakage of the cell's content. During apoptosis, on the other hand, the decrease in forward light scatter (which is a result of the cell shrinkage) is not initially paralleled by a decrease in side scatter. Actually, a transient increase in right angle scatter can be seen during apoptosis in some cell systems (84). This may reflect an increased light reflectiveness by condensed chromatin and fragmented nuclei. In later stages of apoptosis, however, the intensity of light scattered at both, forward and right angle directions, is decreased (84,85).

Cell viability assay by light scatter measurement is simple and can be combined with the analysis of surface immunofluorescence e.g. to identify the phenotype of the dying cell. It can also be combined with functional assays such as mitochondrial potential, exclusion of propidium iodide (PI) or plasma membrane permeability to such dyes as Hoechst 33342 (HO324) or 7-amino actinomycin D (7-AAD), as it will be described further in this article.

It should be stressed, however, that the light scatter changes are not specific to apoptosis. Mechanically broken cells, isolated cell nuclei and necrotic cells also have diminished ability to scatter light. Identification of apoptosis or necrosis by light scatter, therefore, requires additional controls, and should be accompanied by another, more specific assay.


Membrane Permeability Changes. Features distinguishing dead from live cells include loss of transport function and often even the loss of structural integrity of the plasma membrane. Variety of assays of cell viability have been developed based on changes in the properties of the plasma membrane. Because the intact membrane of live cells excludes charged cationic dyes such as trypan blue, PI, ethidium bromide, or 7-AMD, short incubation with these dyes results in selective labeling of dead cells, while live cells show minimal dye uptake (86-92).The assays based on exclusion of these fluorochromes are commonly used to probe cell viability. Generally, a short (5 - 10 min) cell incubation in the presence of these fluorochromes labels dead cells, i.e. the cells that cannot exclude the dye, namely necrotic and late apoptotic cells. The PI exclusion test is frequently used as the flow cytometric equivalent of the trypan blue exclusion assay. These assays can be combined with analysis of cell surface immunophenotype (e.g. 88,91).

During apoptosis the plasma membrane transport function becomes transiently defective prior to total loss of the ability to exclude these charged fluorochromes. At that stage of apoptosis, therefore, the rate of uptake of several of these fluorochromes was shown to be increased, compared to control cells, and it was possible to identify populations of necrotic cells showing intensive fluorochrome labeling after a short incubation with the dye, from the moderately labeled apoptotic cells and from the nonapoptotic, live cells exhibiting minimal fluorochrome uptake (86,88,89).

The fluorochrome ethidium monoazide (EMA) similar to ethidium or propidium iodide, also is a positively charged molecule and is excluded from live and early apoptotic cells. It stains cells which have lost the integrity of their plasma membrane i.e. necrotic and late apoptotic, as well as mechanically damaged cells (93). This dye can be photochemically crosslinked to nucleic acids by exposure to visible light. Cell incubation with EMA, followed by their illumination, irreversibly labels the cells which were unable to exclude the dye during incubation. The photolabeling of EMA can be conveniently combined with membrane immunophenotyping (94).

Another assay of membrane integrity employs the nonfluorescent esterase substrate, fluorescein diacetate (FDA). This substrate, after being taken up by live cells is hydrolyzed by intracellular esterases which are ubiquitous to all types of cells (95). The product of the hydrolysis, fluorescein, is a highly florescent, charged molecule which becomes trapped in intact cells. Incubation of cells in the presence of both propidium iodide (PI) and FDA, thus, labels live cells green (fluorescein) and dead cells red (PI). This is a convenient assay, widely used in flow cytometry.

Another DNA fluorochrome, Hoechst 33342 (HO342), unlike PI, is not excluded by live or apoptotic cells. Actually, it has been observed that short exposure of cells to low concentrations of HO342 led to strong labeling of apoptotic cells (86,96-99), Live cells, on the other hand, required much longer incubation with HO342 to obtain a comparable intensity of fluorescence. Supravital uptake of HO342 combined with exclusion of PI (to identify necrotic and late apoptotic cells) and with analysis of the cell's light scatter properties has been proposed as an assay of apoptosis 86,97). Hoechst fluorochrome HO258 appears to offer an advantage of increased stability of fluorescence, compared to HO342 (100). Other dyes, such as SYTO-16 and LCS-751 can also be used to discriminate apoptotic cells, using the same principle as HO342, offering a choice of fluorochromes with different excitation and emission spectra (101). A combination of cell labeling with HO342 and 7-AMD was shown to discriminate between apoptotic and necrotic cells and to allow one to reveal the surface immunophenotype (90,91).

The degree of change in permeability of the plasma membrane to either charged or uncharged fluorochromes varies with the stage (advancement) of apoptosis, cell type and mode of induction of apoptosis (e.g. DNA damage vs. engagement of the Fas receptor). Therefore, the optimal conditions for discrimination of apoptotic cells (fluorochrome concentration, time and temperature of incubation, often ionic composition of the incubation medium) may significantly vary between different cell systems. Pilot experiments, therefore, are always necessary to customize the conditions for different cell systems for maximal discrimination of apoptotic from live, and/or from necrotic cells.

Detection of Phosphatidylserine on Cell Surface. In live cells plasma membrane phospholipids are asymmetrically distributed between inner and outer leaflets of the plasma membrane. Thus, while phosphatidylcholine and sphingomyelin are exposed on the external surface of the lipid bilayer, phosphatidylserine is located on the inner surface (43). It has been shown recently that loss of phospholipid asymmetry leading to exposure of phosphatidylserine on the outside of the plasma membrane, is an early event of apoptosis (43,102). The anticoagulant annexin V preferentially binds to negatively charged phospholipids such as phosphatidylserine. By conjugating fluorescein to annexin V it has been possible to use such a marker to identify apoptotic cells by flow cytometry (102-104). During apoptosis the cells become reactive with annexin V after the onset of chromatin condensation but prior to the loss of the plasma membrane's ability to exclude PI. Therefore, by staining cells with a combination of fluoresceinated annexin V and PI it is possible to detect nonapoptotic live cells (annexin V negative/PI negative), early apoptotic cells (annexin V positive, PI negative) and late apoptotic or necrotic cells (PI positive) by flow cytometry (103,104).

Loss of F-actin. A rapid loss of plasma membrane structures such as pseudopodia and microvilli, resulting in the smooth appearance of the cell surface under the electron or phase contrast microscope, characterizes changes that occur relatively early during apoptosis (6,8,11). F-actin is a major constituent of pseudopodia. The phallotoxins are toxic cyclic peptides which bind to F-actin and prevent its depolimerization. Fluoresceinated phallotoxins are used as a probe of F-actin. It has been recently reported that the ability of cells to bind fluoresceinated phalloidin is lost during apoptosis (105). It was proposed, therefore, to combine cell staining with fluoresceinated phalloidin with DNA content analysis, to identify apoptotic cells and to reveal the cell cycle position of both, apoptotic and nonapoptotic cell population (105). This approach was tested on HL-60 cells induced to undergo apoptosis by etoposide.


Altered Mitochondrial Transmembrane Potential. Many viability assays are based on functional tests of cell organelles. For example, the charged cationic green fluorochrome rhodamine 123 (Rh123) accumulates in mitochondria of live cells as a result of the transmembrane potential (106). Cell incubation with Rh123 results in labeling of live cells while dead cells, having uncharged mitochondria, show minimal Rh123 retention. Cell incubation with both Rh123 and PI labels live cells green (Rh123) and dead cells red (107). A transient phase of cell death, most likely by necrosis, however, can be detected, when the cells partially loose the ability to exclude PI and yet stain even more intensively than intact cells with Rh123 (107). This suggests that mitochondrial transmembrane potential is transiently elevated early during necrosis, at the time of cell and mitochondrial swelling and prior to rupture of the plasma membrane.

An early event of apoptosis is a decrease in mitochondrial transmembrane potential which is reflected by a loss of the cell ability to accumulate Rh123 or cyanine dyes in mitochondria (108-110). This event is associated with an increased production of superoxide anions (reactive oxygen intermediates) and increased content of the reduced form of cellular glutathione (36,38). The product of bcl-2 gene appears to play a critical role preventing the loss of the mitochondrial transmembrane potential during apoptosis (19-21,36).

Lysosomal Proton Pump. Another organelle which can be probed by flow cytometry are lysosomes. Incubation of cells in the presence of 1-2 µg/ml of the metachromatic fluorochrome acridine orange (AO) results in the uptake of this dye by lysosomes of live cells which fluoresce red (111). The uptake is the result of an active proton pump in lysosomes: the high proton concentration (low pH) causes AO, which can enter the lysosome in an uncharged form, to become protonated and thus entrapped in the organelle. Live cells, thus, especially the cells which have numerous active lysosomes, such as monocytes or macrophages, show strong red- and rather weak green fluorescence. The latter results from AO binding to nucleic acids. At that low AO concentration dead cells exhibit weak green and minimal red fluorescence.

It should be stressed that in the early stages of apoptosis, both the physical integrity of plasma membrane is preserved and lysosomes remain relatively unchanged compared to live cells. The assays based on analysis of the membrane permeability to the dyes (dye exclusion tests), or function of lysosomes, therefore, are not always able to identify early apoptotic cells. However, they are useful to discriminate between live and necrotic or late apoptotic cells.


Changes in chromatin structure during apoptosis are reflected by altered sensitivity of DNA in situ to denaturation. The sensitivity of DNA in situ to denaturation can be measured based on the metachromatic property of the fluorochrome AO. Under proper conditions this dye can differentially stain double stranded (ds) vs. single stranded (ss) nucleic acids (112). Namely, when AO intercalates into ds DNA it emits green fluorescence. In contrast, the products of AO interaction with ss DNA fluoresce red. In this method, the cells are briefly pre-fixed in formaldehyde followed by ethanol postfixation. Cell fixation abolishes staining of lysosomes, as described above in this article. Following removal of RNA from the fixed cells by their preincubation with RNase, DNA is partially denatured in situ by short cell exposure to acid. The cells are then stained with AO at low pH to prevent DNA renaturation. It was shown before that the sensitivity of DNA in situ to denaturation is higher in condensed chromatin of mitotic cells compared to the noncondensed chromatin of interphase cells (112). Apoptotic cells, like mitotic cells, have a larger fraction of DNA in the denatured form, and more intense red- and reduced green- fluorescence, compared to nonapoptotic (interphase) cells; the latter stain strongly green but have low red fluorescence (73,74). An increased degree of DNA denaturation (single-strandedness) in apoptotic cells also can be detected immunocytochemically, using an antibody reactive with ssDNA (113).

The methods based on sensitivity of DNA to undergo denaturation may be uniquely applicable in situations where internucleosomal DNA degradation is not apparent (e.g. 52-54), and thus when other methods of apoptotic cells identification (e.g. the ones that rely on detection of DNA degradation) may fail. They cannot, however, discriminate between mitotic and apoptotic cells. The method utilizing AO has found a practical application to identify abnormal (apoptotic-like) sperm cells from individuals with reduced fertility and to study genotoxic effects of antitumor drugs and environmental poisons during spermatogenesis (e.g. 76,77).


Extensive DNA cleavage, considered to be a hallmark of apoptosis (1-6), provided a basis for the development of two different flow cytometric types of assays to identify apoptotic cells. One relies on extraction of low MW DNA prior to staining and measuring content of cellular DNA (25,114-116). Another type of the assay is based on fluorochrome labeling of DNA strand breaks in situ (26,117-122).

Loss of Cellular DNA. In the first approach, the cellular DNA content is measured following cell permeabilization with detergents or prefixation with precipitating fixatives such as alcohols, or acetone. Cell permeabilization or alcohol fixation does not fully preserve the degraded DNA within apoptotic cells: this fraction of DNA leaks out during subsequent cell rinsing and staining. As a consequence, apoptotic cells contain reduced DNA content and therefore can be recognized, following staining of cellular DNA, as cells with low DNA stainability ("sub-G1"peak), lower that of G1 cells (25,114-116). Furthermore, late apoptotic cells, which lost a portion of their DNA via shedding of apoptotic bodies also are characterized by fractional DNA content.

The degree of DNA degradation varies depending on the stage of apoptosis, cell type and often the nature of the apoptosis-inducing agent. The extractability of DNA during the staining procedure (and thus separation of apoptotic from live cells by this assay), also varies. It has been noted that addition of high molarity phosphate-citrate buffer to the rinsing solution enhances extraction of the degraded DNA (116).This approach can be used to control the extent of DNA extraction from apoptotic cells to the desired level to obtain the optimal separation of apoptotic cells by flow cytometry.

Since measurement of DNA content provides information about the cell cycle position of the nonapoptotic cells, this approach can be applied to investigate the cell cycle specificity of apoptosis. Another advantage of this approach is its simplicity, and applicability to any DNA fluorochrome or instrument. The combination of correlated DNA and RNA measurements, which allows one to identify G0 cells, makes it possible to distinguish whether apoptosis is preferential to G1 or G0 cells (25).

The limitation of the DNA extraction approach is low specificity in the detection of apoptosis. The "sub G1" peak can represent, in addition to apoptotic cells, mechanically damaged cells, cells with lower DNA content (e.g. in a sample containing cell populations with different DNA indices) or cells with different chromatin structure (e.g. cells undergoing erythroid differentiation) in which the accessibility of DNA to the fluorochrome is diminished. As will be discussed further, this is of special concern when unfixed cells are lysed in hypotonic solution, resulting in isolation of multiple nuclear fragments. Hence, the number of "sub G1" cells in such preparation represents the number of nuclear fragments and provides no information on the number of apoptotic cells.

Presence of DNA Strand Breaks. Endonucleolytic DNA cleavage results in extensive DNA breakage. The 3' OH ends in DNA breaks are detected by attaching to them biotin or digoxygenin conjugated nucleotides, in a reaction catalyzed by exogenous TdT ("end-labeling", "tailing", "TUNEL") or DNA polymerase (nick translation) (26,117-122). Fluorochrome conjugated avidin or digoxygenin antibody are then used in the second step of the reaction to label DNA strand breaks. A simplified, single step procedure has been developed, using fluorochromes directly conjugated to deoxynucleotides (122). More recently, a new method was introduced in which BrdUTP, incorporated by TdT, is used as the marker of DNA strand breaks (120,121). The method based on BrdUTP incorporation (122) is simpler, more sensitive and costs less compared with the digoxygenin or biotin labeling. Commercial kits designed to label DNA strand breaks for identification of apoptotic cells are offered by ONCOR Inc., (Gaithersburg, MD, USA; double step assay using digoxygenin) and Phoenix Flow Systems (San Diego, CA, USA, single step and BrdUTP labeling assays).

The approach based on DNA strand break labeling in the assay employing TdT appears to be the most specific in terms of positive identification of apoptotic cells. Namely, necrotic cells or cells with primary breaks induced by ionizing radiation (up to the dose of 25 Gy of radiation), or DNA damaging drugs, have an order of magnitude fewer DNA strand breaks than apoptotic cells (26). Because cellular DNA content of not only nonapoptotic but also apoptotic cells is measured, the method offers a unique possibility to analyze the cell cycle position, and/or DNA ploidy, of apoptotic cells (123).

It has been reported that comparative labeling of DNA strand breaks utilizing DNA polymerase vs. TdT allows one to discriminate between apoptotic and necrotic cells (117). The difference in intensity of labeling of apoptotic and necrotic cells in these assays, however, was inadequate to fully separate these populations (117).

The procedure of DNA strand break labeling is rather complex and involves many reagents. Negative results, therefore, may not necessarily mean the absence of DNA strand breaks but be a result of some methodological problems, such as the loss of TdT activity, degradation of triphosphonucleotides, etc. It is necessary, therefore, to include a positive and negative control. An excellent control consist HL-60 cells treated (during their exponential growth) for 3 - 4 h with 0.2 µM of the DNA topoisomerase I inhibitor camptothecin (CPT). Because CAM induces apoptosis selectively during S phase, the populations of G1 and G2/M cells may serve as negative populations (background), while the S phase cells in the same sample, serve as the positive control.

The detection of apoptotic cells based on the presence of DNA strand breaks can be combined with analysis of DNA replication (121). The advantage of this approach is that it offers a possibility, in a single measurement, to identify the cells which incorporated halogenated DNA precursors and the cells undergoing apoptosis, in relation to their DNA content.


The optimal method for identification of dead cells depends on the cell system, the nature of the inducer of cell death, the mode of cell death, the particular information that is being sought (e.g. specificity of apoptosis with respect to cell cycle phase or DNA ploidy) and the technical restrictions (e.g. the need for sample storage or transportation, type of flow cytometer available, etc.).

The methods based on analysis of plasma membrane integrity (exclusion of charged fluorochromes or FDA hydrolysis), although simple and inexpensive, may fail to identify apoptotic cells, especially at early stages of apoptosis. They can be used to identify necrotic cells, cells damaged mechanically or cells advanced in apoptosis. Their major use is in enumeration of live cells in cultures (e.g. to exclude dead cells during analysis of cell growth curves, as when using the trypan blue exclusion assay), or in discrimination of mechanically broken cells (e.g. following mechanical cell isolation from solid tumors). Careful analysis of the kinetics of cell stainability with these dyes, however, may in some cell systems distinguish necrotic from apoptotic cells.

Positive identification of apoptotic cells is more difficult. The most specific assays appear to be based on the detection of DNA strand breaks. The number of DNA strand breaks in apoptotic cells appears to be of such large magnitude that the intensity of DNA strand break labeling can be a specific marker of these cells (26). The situation, however, is complicated in the case of atypical apoptosis, which, as discussed earlier, may be characterized by the lack of internucleosomal DNA degradation. The number of DNA strand breaks in such atypical apoptotic cells is not always adequate for their identification by this method. The data of Chapman et al., (124), however, indicate that apoptotic cells can be distinguished by DNA the strand breaks assay even in the absence of internucleosomal DNA cleavage. On the other hand false positive recognition of apoptosis, by this method, may occur in situations where internucleosomal DNA cleavage accompanies necrosis.

Apoptosis can be identified with better assurance when more than a single viability assay is used. Thus, simultaneous assessment of plasma membrane integrity (e.g. exclusion of charged fluorochromes or hydrolysis of FDA), together with either membrane permeability (HO342 uptake), mitochondrial transmembrane potential (Rh123 uptake), DNA sensitivity to denaturation or DNA cleavage assays, offer a more certain means of identification of the mode of cell death than each of these methods alone.

Cost and simplicity play a role in choice of the method. The least expensive and most rapid discrimination of apoptotic cells is based on DNA content analysis. This approach is routinely used in our laboratory for screening drug effects in vitro, in particular when large number of samples have to be measured. In addition to the enumeration of apoptotic cells offered by this method, the cell cycle specific effects can easily be recognized from DNA content histograms of the nonapoptotic cell populations. The data are then confirmed using the DNA strand break labeling assay.

Flow cytometry can provide rapid, quantitative and objective assays of cell viability which may be applied for enumeration of apoptotic or necrotic cells. However, regardless of the particular method that has been used to identify the mode of cell death, flow cytometric analysis should always be confirmed by the inspection of cells under the light or electron microscope. Morphological changes during apoptosis are unique and they should be the deciding factor when ambiguity arises regarding the mechanisms of cell death. Furthermore, apoptosis was originally defined based on the analysis of cell morphology (6,7). Morphological criteria of identification of apoptotic and necrotic cells, therefore, should be taken into an account in conjunction with flow cytometric analysis of cell death.


Cell Lysis. One of the common methods of identification of apoptotic cells relies on DNA content analysis: objects with a fractional DNA content are assumed to be apoptotic cells. This, however, holds true for cells which were prefixed with precipitating fixatives such as alcohols or acetone. It is quite common, however, that this analysis is performed on cells which were subjected to treatment with a detergent or hypotonic solution. Such treatment lyses the cell. Because the nucleus becomes fragmented during apoptosis and numerous individual chromatin fragments often are present in a single cell, the percentage of objects with a fractional DNA content (represented by the"sub-G1" peak) released from a lysed cells does not correspond to the apoptotic index. Furthermore, lysis of mitotic cells, or cells with micronuclei releases individual chromosomes or micronuclei, respectively. Individual chromosomes, chromosome aggregates as well as micronuclei are all objects with a fractional DNA content which erroneously can be identified as apoptotic cells.

An exception from the rule of not using detergents is gentle permeabilization of the cell with the detergent but in the presence of exogenous proteins such as serum or serum albumin. The presence of 1% (w/w) albumin or 10% (v/w) serum protects cells from lysis (e.g. induced by 0.1% Triton X-100) without affecting their permeabilization by detergent. In fact, this method is used for simultaneous analysis of DNA and RNA (125). Apoptotic or nonapoptotic cells suspended in saline containing the detergent and serum proteins, however, are fragile and their pipetting, vortexing or even shaking the tube containing the suspension, causes cell lysis and release of the cell constituents into solution.

Erroneous Identification of the Objects with Minimal DNA Content as Apoptotic Cells. It is quite common to see a logarithmic scale (log amplifiers) used to measure and display DNA content when trying to quantitate apoptosis. Such use frequently parallels the use of detergents. A logarithmic scale allows one to measure and record objects with 1% or even 0.1% of the DNA content of intact, nonapoptotic cells. Most of such objects cannot be apoptotic cells. In the case of cell lysis by detergents, as discussed above, these objects represent nuclear fragments, individual apoptotic bodies, chromosomes, chromosome aggregates or micronuclei.

For practical reason it is advisable to identify apoptotic cells as the objects with a fractional DNA content which is no less than 10 % (or 20 %) of that of intact G1 cells. This may result in an underestimate of apoptosis, but the bias is constant and is less of a danger compared to counting all objects with a fractional DNA content, for example ranging from 0.1 % to 100 % of intact G1 cells. In the latter case cellular debris, single chromosomes from broken mitotic cells, chromosome clumps, contaminating bacteria, etc, all having very low DNA content, and may be erroneously classified as apoptotic cells. Application of a linear rather than a logarithmic scale provides better assurance that objects with a minimal DNA content are excluded from the analysis.

Incorrect Assumption that Percent of Apoptotic Cells Represents the Cell Death Rate. Similar to mitosis, apoptosis is of short duration. It is also of variable duration. The time-window during which apoptotic cells demonstrate their characteristic features that allow them to be identifiable varies depending on the method used, cell type or nature of the inducer of apoptosis. Some inducers may slow down or accelerate the apoptotic process by affecting the rate of formation and shedding of apoptotic bodies, endonucleolysis, proteolysis, etc., thus altering the duration of the "time window" by which we identify the apoptotic cell. One has to keep in mind, for example, that an observed two- fold increase in apoptotic index may either indicate that twice as many cells were dying by apoptosis, compared to control, or that the same number of cells were dying but that the duration of apoptosis was prolonged twofold. Unfortunately, no method exists to obtain cumulative estimates of the rate of cell entrance to apoptosis as there is, for example, for mitosis, which can be arrested by microtubule poisons in a stathmokinetic experiment. In short, the percentage of apoptotic cells in a cell population estimated by a given method is not a measure of the rate of the cells dying by apoptosis.

To estimate the rate (kinetics) of cell death the absolute number (not the percent) of live cells should be measured in the culture, together with the rate of cell proliferation. The latter may be obtained from the rate of cell entrance to mitosis (cell "birth rate") in a stathmokinetic experiment. The observed deficit in the actual number of live cells from the expected number of live cells estimated based on the rate of cell birth provides the cumulative measure of cell loss (death). Indirectly, the cell proliferation rate can be inferred from the percentage of cells incorporating BrdUrd or from the mitotic index, under the assumption that the treatment which induces apoptosis does not affect the duration of any particular phase of the cell cycle (generally a risky assumption). The latter approach, however, cannot provide an estimate of the cell birth rate.

Incorrect Assumption that Apoptotic Cells Show All Typical Features of Apoptosis. The lack of evidence of apoptosis, detected by a particular method, is not evidence of the lack of apoptosis. As mentioned, there are examples in the literature where cells die by a process resembling apoptosis which lacks one or more typical apoptotic features. Most frequently, DNA degradation stops after cleavege to 50 - 300 kb fragments and thus fewer in situ DNA strand breaks are present compared with classical apoptosis (35). The method of identification of apoptosis based on detection of the missing feature (e.g. DNA laddering on gels) fails to identify atypical apoptosis in such a situation.

Application of more than a single method, each detecting a different cell feature, provides a better opportunity to detect atypical apoptosis than a single method. One expects, for example, that if DNA in apoptotic cells is fragmented to 50 -300 kb it cannot not be extractable and, as a result, such cells could not be identified as apoptotic either by the method based on analysis of DNA content or that based on "laddering" DNA during electrophoresis. However, the presence of in situ DNA strand breaks, even at lower frequency than in the case of typical apoptosis, can be used as a marker in such cells (124). Furthermore, such apoptotic cells may also be recognized based on their reduced F-actin stainability with FITC-phalloidin (105) or by the FITC-annexin V conjugate reacting with cell surface phosphatidylserine (102).

Mistaken Identification of Late Apoptotic Cells as Necrotic Cells: Cells very advanced in apoptosis resemble necrotic cells to such an extent that the term "apoptotic necrosis" was proposed to define the late stages of apoptosis (8). Because the ability of apoptotic cells to exclude charged cationic fluorochromes such as PI or 7-AMD is lost at these late stages, the discrimination between late apoptosis and necrosis cannot be accomplished by methods utilizing these dyes. Since phosphatidylserine may be accessible to annexin V in necrotic cells due to the fact that the integrity of plasma membrane is compromised, it is expected that this assay also cannot be used to identify apoptotic from necrotic cells. Other methods, therefore, should be used. Extensive DNA fragmentation detected by DNA gel electrophoresis and analysis of cellular DNA content, or in situ presence of numerous DNA strand breaks may serve as markers to distinguish late apoptotic from necrotic cells.

Discard of the Non-attached Cells in Cultures. Relatively early during apoptosis cells detach from the surface of the culture flasks and float in the medium. The standard procedure of discarding the medium, trypsinization or EDTA-treatment of the attached cells and their collection results in selective loss of apoptotic cells. Such loss may vary from flask to flask depending on handling the culture e.g. degree of mixing or shaking, efficiency in discarding the old medium, etc. Surprisingly, cell trypsinization and discarding the medium is quite a common practice in studies of apoptosis. Needless to say, such an approach can not be used for quantitative analysis of apoptosis. To estimate the apoptotic index in cultures of adherent cells, it is essential to pool the floating cells with the tryspsinized ones and measure them together.

Similarly, density gradient (e.g. using ficoll-hypaque solutions) separation of the cells may result in selective loss of dying and dead cells. The knowledge of any selective loss of dead cells in cell populations purified by such approaches is essential when one is studying apoptosis

Use of the Untested Commercial Kits. A number of commercial kits have recently become available to detect apoptosis. Some of these kits have solid experimental foundations and have been successfully tested on a variety of cell systems. Other kits, however, especially those advertised by vendors who do not fully explain the principle of detection of apoptosis on which the kit is based, and do not list its chemical composition, may not be universally applicable. Unfortunately, it is a common practice that some manufacturers have tested only a single cell line using a single agent to trigger apoptosis (generally, either a leukemic cell line treated with the FAS ligand, or HL-60 cells treated with a DNA topoisomerase inhibitor). Yet the claim is often made that their kit is applicable to different cell systems. Before application of any new kit it is advisable to confirm that at least 3-4 independent laboratories have already successfully used it on different cell types. Furthermore, it is good practice to use the new kit in parallel with an well established methodology, in a few experiments. This would allow one, by comparison of the apoptotic indices, to estimate the time- window of detection of apoptosis by the new method, compared to the one which is already established and accepted in the field.

Neglect to Examine the Cells by Microscopy. Apoptosis was originally defined as specific mode of cell death based on very characteristic changes in cell morphology (7). Although individual features of apoptosis may serve as markers for detection and analysis of the proportion of apoptotic cells in the cell populations studied by flow cytometry, the mode of cell death always should be positively identified by inspection of cells by light or electron microscopy. Therefore, when analysis is done by flow cytometry and any ambiguity arises regarding the mechanism of cell death, the morphological changes should be the deciding attribute in resolving the uncertainty.

It should be stressed that optimal preparations for light microscopy require cytospining of live cells following by their fixation and staining on slides. The cells are then flat and their morphology is easy to assess. On the other hand, when the cells are initially fixed and stained in suspension, then transferred to slides and analyzed under the microscope, their morphology is obscured by the unfavorable geometry: the cells are spherical and thick and require confocal microscopy to reveal details such as early signs of apoptotic chromatin condensation.

Differential staining of cellular DNA and protein with DAPI and sulforhodamine 101 of the cells on slides, which is very rapid and simple, gives a very good morphological resolution of apoptosis and necrosis (5). Other DNA fluorochromes, such as PI or 7-AMD, or the DNA/RNA fluorochrome AO, can be used as well.


The explosive growth in recent years of flow cytometry for the analysis of cell death in a variety of disciplines of biology and medicine is the best evidence of the value of this methodology in cell necrobiology. Two general directions characterize this growth. One direction is the use of flow cytometry to quantify apoptotic cells (apoptotic index). Compared to the alternative methods (analysis of cell morphology, DNA gel electrophoresis) flow cytometry is rapid, objective and very sensitive. As mentioned, however, since identification of apoptotic cells by flow cytometry is generally based on a single feature, which may not necessarily be the marker of apoptosis in every situation, the mode of cell death should be confirmed by light or electron microscopy.

Flow cytometric methods to quantify apoptotic cells have already found application in clinical oncology. Tumor apoptotic index prior to treatment appears to have predictive value, at least in some tumor types (67). Clearly, the knowledge of the rate of cell proliferation and cell death is more predictive of the rate of tumor growth than information on the rate of cell proliferation alone. Furthermore, analyzing the rate of death of tumor cells during treatment offers a unique possibility to assess the efficiency of the treatment very early, before other clinical parameters of treatment efficiency can be measured. Such an application revealed the kinetics of cell death in various types of leukemia treated with different drugs (32,66,67). There is no adequate evidence yet to conclude whether the assessment of the apoptotic index during treatment has long term prognostic value, but with some drugs, (e.g. the DNA topoisomerase I inhibitor Topotecan or microtubule poison Paclitaxel) apoptosis appears to correlate well with the initial clinical response (67). Another clinical application of these methods where apoptosis also appears to be of predictive value is in the analysis of spontaneous- or activation induced-apoptosis of lymphocytes in the course of HIV infection (71). These routine clinical applications call for development of standardized procedures and for quality control that could be generally accepted in clinical practice.

The second area of cell necrobiology where flow cytometry is already widely applied is in the study of molecular mechanisms associated with cell death. In this area, flow cytometry offers unique opportunities and is even more advantageous compared to alternative techniques. By virtue of the possibility of correlating measurements of multiple parameters on the same cells, the methodology allows one to detect cells undergoing apoptosis with respect to their cell cycle position, without the need for cell synchronization, or to study the relationship between apoptosis and a particular function of the cell or cellular organelle. Multiparametric analysis combining immunocytochemical detection of individual proteins with apoptotic markers and with DNA content analysis is a powerful approach which can be used to relate expression of individual genes to cell cycle position and the cell's propensity to undergo apoptosis. This allows one to study interactions between components of the regulatory machinery of the cell cycle and apoptosis, mechanism of action of cytotoxic drugs and the effects of biological modifiers on target cells and other molecular interactions associated with cell death. Needless to say, the molecules directly related to the regulatory mechanisms of apoptosis, such as proteins of the bcl-2 and ICE families, the status of c-myc or ras proteins, or the expression of the tumor suppressor p53, can be directly measured and correlated with each other, or with still other cell components related to cell death, proliferation or differentiation. One may expect that flow cytometry will contribute to the progress in cell necrobiology as much, or even more, as it did to the progress of research on cell proliferation and the cell cycle.

Supported by NCI Grant RO1 CA 28 704, the Chemotherapy Foundation and "This Close" for Cancer Research Foundation.


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