Flow cytometry of apoptotic cell death
Introduction
The term apoptosis or programmed cell death (PCD) defines a genetically encoded cell death program, which is morphologically, biochemically and molecularly distinct from necrosis. Apoptosis is distinguished from necrosis by unique events including the degradation of chromatin into internucleosomal fragments, a loss of cellular volume associated with cytoskeletal breakdown and blebbing of the plasma membrane. Apoptotic and necrotic cell death are different from a mechanistic point of view because necrosis is merely the passive result of cellular injury and apoptosis forms an integral part of normal physiological cell processes. Apoptosis ensures an equilibrium between cell proliferation and cell death and plays a regulatory role in the control of the size of cell populations and tissues (Kerr et al., 1972). Continuous signalling by growth factors, hormones, cytokines, cell–cell contacts and cell–matrix interactions are necessary for cells to refrain from undergoing apoptosis, keeping them alive. Cells which are most sensitive for survival signals stay alive and cells that can not compete with their more avid sister cells undergo apoptosis due to relative shortness of survival factors. Aberrations in cell death signalling, in membrane or cytoplasmic receptors or alterations in genes that govern apoptosis are involved in the pathogenesis of congenital malformations and many acquired diseases (Haanen and Vermes, 1996). Too little apoptosis may result in malignancies (Sturzbecher et al., 1990, Tomlinson and Bodmer, 1995), lymphoproliferative diseases (Nagata and Golstein, 1995), leukemias (Mapara et al., 1993, Lepelley et al., 1995, Sachs, 1996, Estrov and Talpaz, 1996), resistance to anticancer therapy (Fisher, 1994, D’Amico and McKenna, 1994, Pahor et al., 1996), persistence of viral infections (Chiou et al., 1994, Haanen and Vermes, 1995) or the occurrence of autoimmune diseases (Nagata and Suda, 1995, Tax et al., 1995). Too much apoptosis can result in immune deficiency (Groux et al., 1991, Ameisen, 1992, Meyaard et al., 1992, Kabelitz et al., 1993, TeVelde et al., 1996) and degenerative conditions (Griffith et al., 1995). It is therefore important to discriminate between necrosis and apoptosis in order to learn how to modulate apoptosis in view of its potential therapeutic use.
The very nature of the apoptotic cell death promotes the underestimation of this phenomenon, because apoptosis involves scattered single cells of which the early stages of the apoptotic process escape recognition, the apoptotic bodies are small and undergo rapid phagocytosis. The duration of the whole process takes not more than a few hours and finally, any inflammatory reaction is absent (Wyllie et al., 1980, Trump et al., 1982).
The various cell biological alterations, which occur during the process of apoptosis take place in an orderly sequence. A number of these can be exploited to discriminate vital and dying cells and to analyse the extent and the type of cell death (apoptosis or necrosis). In this review those cellular changes, which can be measured by flow cytometry (FCM), are discussed according to the sequence at which they occur. Apoptosis starts with cell shrinkage, expressed by changes in cell light-scatter (2) signals. Initially the integrity of the cell membrane (3) remains intact. Activation of caspases (4), leads to the transition of the mitochondrial membrane potential (5), accompanied by intracellular shifts in Ca2+ and pH (6). After loss of the mitochondrial membrane potential, the lysosomal membrane pumps (7) lose their function. At this stage, when the cell has passed the point of no return, endogenous endonucleases are activated, which is reflected by phosphatidylserine redistribution (11). Finally the cell disintegrates into apoptotic bodies (12) (Fig. 1). Detailed descriptions of cell features, which can be measured by FCM during apoptosis have recently been given in comprehensive reviews by Darzynkiewicz et al. (Darzynkiewicz et al., 1992, Darzynkiewicz et al., 1994, Darzynkiewicz et al., 1997), Telford et al. (1994), Vermes and Haanen (1994), Vermes et al. (1998), Gorczyca et al. (1998) and Robinson et al. (1998).
Section snippets
FCM of cell light-scatter
In most but not all cases apoptosis can be distinguished from necrosis on the basis of the scatter parameters as measured by FCM (Ormerod et al., 1995). The forward-angle light scatter (FSC) relates to the cell diameter, the side-angle light scatter (SSC) reflects the conformation of inner cellular structures. During the initial stages of apoptosis the cell shrinks, while the membrane remains intact. During necrosis cell swelling occurs as a result of the early failure of the membrane
FCM of dye uptake
FCM can be utilized for quantitative analysis of the number of vital, apoptotic and necrotic cells by the rate of uptake and retention of certain dyes. Apoptosis is characterized by a maintenance of an intact plasma membrane during a significant part of its time course. The intact plasma membrane integrity includes preservation of its basal function, like a barrier for ions and macromolecular structures, and active transport pumps (Wyllie et al., 1980, Vermes et al., 1998). Relying on
FCM of caspases
Apoptosis was recognized as death by an orchestrated sequence of countless cuts by hydrolytic enzymes, which degrade macromolecular structures like DNA and cytoskeleton, which underlie the observed apoptotic morphology (Martin and Green, 1995). A directing role in this hydrolytic eruption is played by members of a family of cysteine proteases, bearing an active site with a conserved amino acid sequence and which cleave specifically following aspartate residues (Kumar and Lavin, 1996). These
FCM of mitochondrial function
The mitochondrion has been suggested to be fundamental to the biochemistry of apoptosis for it might form the nidus where the decision of life and death is actually being made (Kroemer et al., 1997, Green and Koemer, 1998, Green and Reed, 1998). A crucial event in the role of the mitochondrion is the formation of permeability transition pores (mitochondrial megachannel) in its outer membrane leaflet allowing mitochondrial proteins to flux into the cytosol (Marchetti et al., 1996, Susin et al.,
FCM of calcium flux and pH
Elevation in the cytosolic Ca2+ level and a selective loss of pH regulation resulting in intracellular acidification are events of the apoptotic process. Energy dependent Ca2+ transport systems maintain the cytosolic Ca2+ concentration at 100 nM, at least four orders of magnitude below that found in the extracellular milieu under physiological conditions. In addition to its established role in mediating the effects of mitotic stimuli, elevation in the cytosolic Ca2+ concentration is also
FCM of lysosomal proton pump
Acridine orange (AO) is a fluorescent dye, which easily traverses the cell membrane. Because of its basic property, it is accumulated in lysosomes, which due to an ATP-dependent proton pump, have a low pH inside. Once inside, the dye is protonated and becomes entrapped in these organelles. The supravital cell staining with a low concentration AO (1–5 μM), which appears as red fluorescence, is the reflection of the activity of the proton pump of lysosomes (Darzynkiewicz and Kapuscinski, 1990,
FCM of DNA strand breaks
Activation of the apoptosis-associated endonuclease (caspase-activated DNase, CAD) results in extensive DNA cleavage and thus generates a large number of DNA strand breaks (Enari et al., 1998). Cleavage of the DNA may yield double-stranded, low molecular weight DNA fragments (mono- and oligonucleosomes) as well as single stranded breaks (‘nicks’) in high molecular weight DNA. The presence of 3′hydroxyl-termini of the strand breaks can be detected by labelling with modified nucleotides (e.g.
FCM of cellular DNA content
When thymocytes are incubated with dexamethasone or cell cultures are exposed to cytotoxic drugs at concentrations which elicit apoptosis, DNA-FCM with propidium iodide (PI) reveals a sub-population of cells, designated A0 cells, with reduced DNA stainability. The peak is below the normal G0/G1 region, (Nicoletti et al., 1991). It is believed that the reduced DNA stainability is the consequence of progressive loss of DNA from the cells, due to activation of endogenous endonuclease and
FCM of protein and RNA content
Apoptotic cells in addition to a lowered DNA content show also a diminished protein content (Darzynkiewicz et al., 1992). The lowering of cellular protein level occurs simultaneously with the decrease in DNA content. Circumstantial evidence suggests that proteolysis is necessary for DNA degradation to occur during the apoptotic process. Because the plasma membrane of necrotic cells is leaky, the protein content of these cells is also reduced. RNA in growing and protein synthezising cells is
FCM of phospholipid redistribution
A change of the architecture of the plasma membrane during apoptosis involves the redistribution of the various phospholipid species between the two leaflets of the membrane. Under viable conditions the cell maintains lipid asymmetry over these two leaflets. The most pronounced feature of this asymmetry is the almost complete absence of phosphatidylserine (PS) in the outer leaflet of the plasma membrane. Such steady state situation arises from activities which translocate PS from the outer to
FCM of apoptotic bodies
If the decision to die has been made and the point of no return is passed a series of biochemical alterations occur: degradation of the intracellular structures, crosslinking of proteins and alterations of membrane properties resulting in blebbing and finally in formation of apoptotic bodies (Kerr et al., 1972, Wyllie et al., 1980, Vermes and Haanen, 1994). Biochemical analysis of these small vesicles showed that they consist of highly crosslinked protein envelopes which are rather resistant to
Concluding remarks: choice of technique
FCM allows analysis of cells in suspension, one at a time, at rates of 1000 to 10 000 cells/s. It provides quantitative data about distributions of a wide choice of parameters, ranging from simple cell sizing to measures of cell membrane properties, cytoplasmic constituents, cell organelles, DNA content and nuclear chromatin. All described flow cytometric apoptosis assays have a solid experimental basis and have been used successfully in a variety of cell systems. The restriction of FCM assays
References (127)
Programmed cell death and AIDS: From hypothesis to experiment
Immunol. Today
(1992)- et al.
Binding of vascular anticoagulant (VACa) to planar phospholipid bilayers
J. Biol. Chem.
(1990) - et al.
Exposure of phosphatidylserine in the outer leaflet of human red cells. Relationship to cell density, cell age and clearance by mononuclear cells
J. Biol. Chem.
(1994) - et al.
Apoptosis and a re-investigation of the biological basis for cancer therapy
Radiother. Oncol.
(1994) - et al.
Assays of cell viability: discrimination of cells dying by apoptosis
Methods Cell. Biol.
(1994) - et al.
The S-phase cytotoxicity of camptothecin
Exp. Cell. Res. (March)
(1991) - et al.
The S-phase cytotoxicity of campothecin
Exp. Cell Res.
(1991) - et al.
An adherent cell model to study different stages of apoptosis
Exp. Cell Res.
(1995) Phospholipid flippases
FEBS Lett.
(1988)- et al.
Analysis and discrimination of necrosis and apoptosis (programmed cell death) by multiparameter flow cytometry
Biochim. Biophys. Acta
(1992)
Comparison of cellular and nuclear flow cytometric techniques for discriminating apoptotic subpopulations
Exp. Cell Res.
Induction and activation of tissue transglutaminase during programmed cell death
FASEB Lett.
Apoptosis in cancer therapy: Crossing the threshold
Cell
Detection of DNA damage in individual cells by flow cytometric analysis using anti-ssDNA monoclonal antibody
Exp. Cell Res.
Monoclonal antibody to single-stranded DNA is a specific and sensitive cellular marker of apoptosis
Exp. Cell Res.
In situ detection of fragmented DNA (TUNEL assay) fails to discriminate among apoptosis, necrosis and autoloytic cell death: a cautionary note
Hepatology
Apototic pathays: The roads to ruin
Cell
The central executioners of apoptosis: caspases or mitochonria?
Cell. Biol.
A new generation of Ca2+ indicators with greatly improved fluorescence properties
J. Biol. Chem.
Apoptosis: Programmed cell death in fetal development
Eur. J. Obstetr. Gynecol.
Use of intracellular pH and annexin-V flow cytometric assay to monitor apoptosis and its suppression by Bcl-2 overexpression in hybridoma cell culture
J. Immunol. Methods
Activation-induced cell death (apoptosis) of mature peripheral lymphocytes
Immunol. Today
Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis
Blood
Mitochondrial control of apoptosis
Immunol. Today
Protease activation during apoptosis: death by a thousand cuts?
Cell
Fas and Fas ligand: lpr and gld mutations
Immunol. Today
A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry
J. Immunol. Methods
Protein kinase-C involvement in thymocyte apoptosis induced by hydrocortisone
Cell Immunol.
Calcium-channell blockade and incidence of cancer in aged populations
Lancet
The effect of the cyclin-dependent kinase inhibitor Olomoucine on cell cycle kinetics
Exp. Cell Res.
Caspases: preparation and characterization
Methods: A Companion to Methods in Enzymology
Flow cytometry and biochemical analysis of DNA degradation characteristic of two types of cell death
FASEB Lett.
The use of flow cytometry for the investigation of cell death
Cytometry
Apoptosis: The role of endonuclease
Am. J. Pathol.
Exposure of endogenous phosphatidylserine at the outer surface of stimulated platelets is reversed by restoration of aminophospholipid translocase activity
Biochemistry
Expression of apoptosis-controlling proteins in acute leukemia cells
Leuk. Lymphoma
Sequential acquisition of mitochondrial and plasma membrane alterations during early lymphocyte apoptosis
J. Immunol.
Caspase cleavage of keratin 18 and reorganization of intermediate filaments during epithelial cell apoptosis
J. Cell Biol.
A cautionary note on the use of the TUNEL stain to determine apoptosis
Mol. Neurosci.
Functional complementation of the adenovirus E1B 19-kilodalton protein with Bcl-2 in the inhibition of apoptosis in infected cells
J. Virol.
In vitro generation of endothelial microparticles and possible prothrombotic activity in patients with lupus anticoagulant
J. Clin. Invest.
Analysis of glucocorticoid actions on rat thymocyte deoxyribonucleic acid by fluorescence-activated flow cytometry
Endocrinology
Microfluorometric investigations of chromatin structure
Histochemistry
Proteases to die for
Genes Dev.
Annexin V as a probe of aminophospholipid exposure and platelet membrane vesiculation: a flow cytometry study showing a role for free sulfhydryl groups
Blood
Measurements of cell death by flow cytometry
Acridine orange: A versatile probe of nucleic acids and other cell constituents
Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis)
Cytometry
Features of apoptotic cells measured by flow cytometry
Cytometry
Role of translocases in the generation of phosphatidylserine asymmetry
J. Memb. Biol.
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