Identification of apoptotic cells by formamide-induced dna denaturation in condensed chromatin.

In this article we describe a novel effect of formamide on DNA of apoptotic nuclei and present a method for specific detection of apoptotic cells based on this effect. Our observations show that formamide induces DNA denaturation in apoptotic nuclei but has no such effect on DNA of non-apoptotic cells. Formamide-induced DNA denaturation combined with detection of denatured DNA with a monoclonal antibody (MAb) against single-stranded DNA made it possible to specifically identify the apoptotic cells. This procedure produced intense staining of the condensed chromatin in the apoptotic nuclei. In contrast, necrotic cells from cultures treated with sodium azide, saponin, or hyperthermia did not bind this antibody, demonstrating the specificity of the formamide-MAb assay for the apoptotic cells. However, TUNEL stained 90-100% of necrotic cells in all three models of necrosis. Because the MAb did not stain cells with single- or double-stranded DNA breaks in the absence of apoptosis, we conclude that staining of the apoptotic nuclei is not influenced by DNA breaks and is induced by specific changes in condensed chromatin, such as damage to the DNA-histone interactions. Importantly, the formamide-MAb technique identified apoptotic cells in frozen sections and in histological sections of formalin-fixed, paraffin-embedded tissues.     

DNA localization in nuclear fragments of apoptotic ameloblasts using anti-DNA immunoelectron microscopy: Programmed cell death of ameloblasts

Ameloblasts responsible for tooth enamel formation are classified into two different phases: secretion and maturation. At the transition between these secretion and maturation stages, a considerable number of cells die. In this study, we examined the morphology of degenerating ameloblasts by conventional electron microscopy, and DNA cleavage in degenerating ameloblast nuclei by the in situ terminal transferase assay. The results suggest that apoptosis (programmed cell death) in ameloblasts, including DNA ligation is induced at the transitional stage. The nuclear fragments, chromatin condensation and DNA relocation in apoptotic nuclei were examined quantitatively by post-embedding anti-DNA immunogold electron microscopy and the in situ terminal transferase assay combined with electron microscopy. Numerical analysis revealed that immunogold labeling density in the condensed chromatin of apoptotic nuclei was comparable on the average to that in the perinuclear heterochromatin of normal nuclei, and that individual apoptotic nuclear fragments exhibited highly variable gold particle density, from fragments with lower density to that of normal heterochromatin, to fragments with densities twice as high as that of normal heterochromatin. The in situ terminal transferase assay combined with electron microscopy detected DNA ends exposed by ultrathin sectioning as well as DNA cleavage by a putative endonuclease. In conclusion, the state of the DNA, including its ligation and degeneration, changes gradually during chromatin condensation and nuclear fragmentation of apoptosis.     

Comparison of comet assay, electron microscopy, and flow cytometry for detection of apoptosis.

Differentiating apoptosis from necrosis is a challenge in single cells and in parenchymal tissues. The techniques available, including in situ TUNEL (Terminal deoxyribonucleotide transferase-mediated dUTP-X Nick End-Labeling) staining, DNA ladder assay, and flow cytometry, suffer from low sensitivity or from a high false-positive rate. This study, using a Jurkat cell model, initially evaluated the specificity of the neutral comet assay and flow cytometry compared to the gold standard, electron microscopy, for detection of apoptosis and necrosis. Neutral comet assay distinguished apoptosis from necrosis in Jurkat cells, as evidenced by the increased comet score in apoptotic cells and the almost zero comet score in necrotic cells. These findings were consistent with those of electron microscopy and flow cytometry. Furthermore, using rats with burn or ischemia/reperfusion injury, well-established models of skeletal and cardiac muscle tissue apoptosis, respectively, we applied the comet assay to detect apoptosis in these muscles. Neutral comet assay was able to detect apoptotic changes in both models. In the muscle samples from rats with burn or ischemia-reperfusion injury, the comet score was higher than that of muscle samples from their respective controls. These studies confirm the consistency of the comet assay for detection of apoptosis in single cells and provide evidence for its applicability as an additional method to detect apoptosis in parenchymal cells.     

褐飞虱若虫中肠凋亡细胞的检测

为揭示褐飞虱Niloparvata lugens Stl若虫在发育过程中中肠的凋亡细胞,使用末端脱氧核苷酸转移酶介导的dUTP-生物素断端标记法(TUNEL)进行中肠组织切片检测,结果表明,1～5龄若虫中肠分别存在2%～5%的凋亡细胞。利用4′,6-二脒基-2-苯基吲哚二盐酸(DAPI)染色法检测表明,存在Ⅰ,Ⅱa和Ⅱb期凋亡的细胞核,其特征包括染色体浓缩、边缘化及细胞核碎裂。透射电子显微镜检测结果表明,早期凋亡的细胞呈现染色质浓缩、边缘化特征,晚期凋亡的细胞出现细胞核碎裂、形成凋亡小体及细胞质空泡化等。本研究揭示了在正常发育过程中褐飞虱若虫中肠有少量的细胞发生了凋亡。通过人工干预的方式调控中肠细胞的凋亡进程有可能使之成为防治该水稻害虫的新靶标。     

用TUNEL法检测植物原生质体的调亡

ＴＵＮＥＬ是近年来发展的一种凋亡细胞进行原位检测的方法,可以特异性地标记完整的凋亡细胞核或凋亡小体的染色体３’－ＯＨ断裂末端,但在植物细胞中的应用还不多。本文报道应用ＴＵＮＥＬ法检测胡萝卜原生质体的凋亡,并与ＤＮＡ电泳、彗星电泳等方法进行了比较,结果表明它是一种适用于植物原生质体凋亡检测的灵敏度较高的方法。     

用TUNEL法检测植物原生质体的凋亡

TUNEL是近年来发展的一种对凋亡细胞进行原位检测的方法,可以特异性地标记完整的凋亡细胞核或凋亡小体的染色体3'_OH断裂末端,但在植物细胞中的应用还不多。本文报道应用TUNEL法检测胡萝卜原生质体的凋亡,并与DNA电泳、彗星电泳等方法进行了比较,结果表明它是一种适用于植物原生质体凋亡检测的灵敏度较高的方法。     

Staining apoptosis in paraffin sections. Advantages and limits

OBJECTIVE: To describe the advantages and limits of apoptosis detection on paraffin sections by TdT-mediated dUTP nick end labeling (TUNEL). STUDY DESIGN: Two hundred sixty-five paraffin-embedded samples from malignant and benign human tissue were analyzed by TUNEL. Also, biparametric analysis of apoptosis and proliferation index (MIB-1), apoptosis, cytokeratin or leukocyte common antigen was performed. RESULTS: Our preliminary conclusions are as follows. The limits are that this labelling method might detect cells that have not shown DNA fragmentation specific for apoptosis only. The technique is extremely sensitive to the degree of proteolytic digestion. TUNEL identifies nuclei in areas of necrosis. Indeed, the staining of necrotic areas of tissue with the in situ labelling method should not cause confusion since simple morphologic examination of tissues will suffice to identify areas of necrotic cells. The advantages are that TUNEL is a method of simplifying the identification of apoptotic nuclei in routinely processed tissue sections, maintaining topography. It allows retrospective studies and biparametric analysis of cell death and proliferation on the same sample. Furthermore, with biparametric stain, it could better identify the origin (epithelial, mesenchymal, and so on) of apoptotic cells. CONCLUSION: TUNEL is a good method of detecting apoptotic nuclei in fixed, embedded tissue sections, but, because of the limits of the method, the results should be interpreted in conjunction with apoptosis assessment by routine light microscopy.     

On the use of an appropriate TdT-mediated dUTP–biotin nick end labeling assay to identify apoptotic cells

Apoptosis is an essential cellular mechanism involved in many processes such as embryogenesis, metamorphosis, and tissue homeostasis. DNA fragmentation is one of the key markers of this form of cell death. DNA fragmentation is executed by endogenous endonucleases such as caspase-activated DNase (CAD) in caspase-dependent apoptosis. The TUNEL (TdT-mediated dUTP–biotin nick end labeling) technique is the most widely used method to identify apoptotic cells in a tissue or culture and to assess drug toxicity. It is based on the detection of 3′-OH termini that are labeled with dUTP by the terminal deoxynucleotidyl transferase. Although the test is very reliable and sensitive in caspase-dependent apoptosis, it is completely useless when cell death is mediated by pathways involving DNA degradation that generates 3′-P ends as in the LEI/L-DNase II pathway. Here, we propose a modification in the TUNEL protocol consisting of a dephosphorylation step prior to the TUNEL labeling. This allows the detection of both types of DNA breaks induced during apoptosis caspase-dependent and independent pathways, avoiding underestimating the cell death induced by the treatment of interest.     

Detection of DNA damage in prokaryotes by terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling

Numerous agents can damage the DNA of prokaryotes in the environment (e.g., reactive oxygen species, irradiation, and secondary metabolites such as antibiotics, enzymes, starvation, etc.). The large number of potential DNA-damaging agents, as well as their diverse modes of action, precludes a simple test of DNA damage based on detection of nucleic acid breakdown products. In this study, free 3'-OH DNA ends, produced by either direct damage or excision DNA repair, were used to assess DNA damage. Terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) is a procedure in which 3'-OH DNA ends are enzymatically labeled with dUTP-fluorescein isothiocyanate using TdT. Cells labeled by this method can be detected using fluorescence microscopy or flow cytometry. TUNEL was used to measure hydrogen peroxide-induced DNA damage in the archaeon Haloferax volcanii and the bacterium Escherichia coli. DNA repair systems were implicated in the hydrogen peroxide-dependent generation of 3'-OH DNA ends by the finding that the protein synthesis inhibitors chloramphenicol and diphtheria toxin blocked TUNEL labeling of E. coli and H. volcanii, respectively. DNA damage induced by UV light and bacteriophage infection was also measured using TUNEL. This methodology should be useful in applications where DNA damage and repair are of interest, including mutant screening and monitoring of DNA damage in the environment.     

Flow cytometric scoring of apoptosis compared to electron microscopy in gamma irradiated lymphocytes

One of the early events occurring at the cell membrane during apoptosis is the translocation of phosphatidylserine from the inner side of the plasma membrane to the outer layer. These phosphatidylserine groups can be bound by fluorescein isothiocyanate (FITC)-labelled annexin V. The aim of this study was to evaluate the power of the annexin V flow cytometric assay in detecting apoptosis in gamma irradiated peripheral blood lymphocytes and in differentiating between apoptosis and primary necrosis in these cells. Therefore, 5 Gy and 20 Gy gamma irradiated peripheral blood mononuclear cells (PBMCs) were examined after a 24-h culture period. The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) technique was performed as well. A comparison with an electron microscopic (EM) evaluation was made. EM is based on established morphological criteria allowing the classification of cells into four groups: viable, early apoptotic, secondary necrotic and primary necrotic cells. EM performed on annexin V positive sorted cells proved that a 5 Gy gamma irradiation of PBMCs mainly causes apoptosis, whereas a 20 Gy gamma irradiation mainly induces primary necrosis. Neither the annexin V flow cytometric assay nor the TUNEL assay were able to distinguish between primary and secondary necrotic cells. These results illustrate that if quantification of apoptosis is required, one should be careful in interpreting flow cytometric results obtained by annexin V or TUNEL staining in peripheral blood lymphocytes. Although in general primary necrotic cells show an increased forward scatter due to cellullar swelling, both early apoptotic and necrotic (primary or secondary) lymphocytes show a decreased forward scatter signal. Moreover, both primary and secondary necrotic lymphocytes are annexin V and propidium iodide (PI) positive and therefore indistinguishable. We conclude that if a new experiment focusing on apoptosis is set up, an initial EM evaluation is mandatory. If EM shows that the apoptosis inducing agent used in the design of the experiments is not causing primary necrosis, than the annexin V flow cytometric assay can provide rapid and quantitative information about apoptosis.     

Early detection of staurosporine-induced apoptosis by comet and annexin V assays

Comet, TUNEL, and annexin V assays were used to identify DNA fragmentation and plasma membrane alterations occurring during staurosporine-induced apoptosis in Chinese hamster ovary cells. TUNEL assay detected apoptotic cells after 6 h treatment. The occurrence of annexin V immunofluorescence staining after 1 h treatment confirms that exposure of phosphatidylserine (PS) residues is an early biochemical feature of apoptosis. According to intensity, three annexin staining patterns were distinguished, related to different steps in the apoptotic process. The detection of highly damaged cells by the comet assay after 3 h treatment occurred earlier than the detection of DNA modifications by the TUNEL assay, but later than the exposure of PS residues. However, late apoptotic cells, otherwise characterized by plasma membrane disruption and high annexin V staining, were not detected by the comet assay. In this case, comet assay modified by omitting electrophoresis (halo assay) was more sensitive for an accurate quantification of the apoptotic fraction. Accepted: 2 June 1999     

Evidence for apoptosis correlated with mortality in the giant black tiger shrimp Penaeus monodon infected with yellow head virus

Histological, cytochemical and ultrastructural changes in giant black tiger shrimp Penaeus monodon were investigated at various time intervals after injection with yellow head virus (YHV). Hemocytes, lymphoid organs (LO) and gills were the main focus of the study. After injection with YHV, onset of mortality varied from 36 h onward. By normal hematoxylin and eosin staining, the 3 tissues showed clear and increasing prevalence of nuclear condensation, pyknosis and karyorrhexis from approximately 36 h post-injection (p.i.) until death, although pathology was evident in the LO as early as 12 h p.i. in some shrimp. By nuclear DNA staining with 4',6-diamidino-2-phenylindole (DAPI) and by specific labeling of 3'-OH ends of nuclear DNA using a technique called terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick-end labeling (TUNEL), cells of the 3 tissues showed evidence of chromatin condensation and DNA fragmentation, respectively. Both are generally considered to be characteristic of apoptosis. In addition to TUNEL labeling, evidence for DNA fragmentation was supported by the appearance of approximately 200 base pair DNA ladders at approximately 48 h p.i. in hemocytes of YHV-infected but not uninfected shrimp. Transmission electron microscopy (TEM) of LO tissue revealed features of apoptosis in tissues of YHV-infected shrimp only. These included marginated, condensed and fragmented chromatin without concurrent cytoplasmic damage. Histological, cytochemical, ultrastructural and biochemical data were consistent with the hypothesis that widespread and progressive apoptosis occurred in susceptible shrimp infected with YHV. Although no specific tests were carried out to determine whether this purported apoptosis was the cause of mortality, moribund shrimp had extensive deterioration of vital tissues such as the hemolymph, gills, heart and LO, suggesting that many essential bodily functions had been severely compromised. This probably resulted in the gross signs of lethargy and weakness seen, and it is reasonable to suggest that further, progressive deterioration could have led to the collapse of vital functions followed by death.     

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently attach a tagged form of dUTP to 3’ ends of double- and single-stranded DNA breaks in cells. It is a reliable and useful method to detect DNA damage and cell death in situ. This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method of semi-automated TUNEL signal quantitation. Inherent normal tissue features and tissue processing conditions affect the ability of the TdT enzyme to efficiently label DNA. Tissue processing may also add undesirable autofluorescence that will interfere with TUNEL signal detection. Therefore, it is important to empirically determine tissue processing and TUNEL labeling methods that will yield the optimal signal-to-noise ratio for subsequent quantitation. The fluorescence-based assay described here provides a way to exclude autofluorescent signal by digital channel subtraction. The TUNEL assay, used with appropriate tissue processing techniques and controls, is a relatively fast, reproducible, quantitative method for detecting apoptosis in tissue. It can be used to confirm DNA damage and apoptosis as pathological mechanisms, to identify affected cell types, and to assess the efficacy of therapeutic treatments in vivo.     

Noninvasive Monitoring of Apoptosis versus Necrosis in a Neuroblastoma Cell Line Expressing a Nuclear Pore Protein Tagged with the Green Fluorescent Protein

A fusion chimera between the integral nuclear pore membrane protein POM121 and GFP (green fluorescent protein) has been shown to correctly target to the nuclear pores when transiently expressed in a number of mammalian cell types. POM121-GFP is therefore an excellent marker for the noninvasive studies of the nuclear pores in living cells using fluorescence microscopy. We have established a line of neuroblastoma cells stably expressing the POM121-GFP fusion protein. We also monitored the nuclear envelope in living cells after induction of apoptosis or necrosis using 1 μM staurosporine or 100 μMp-benzoquinone, respectively. Interestingly, the POM121-GFP fluorescence was weaker or missing in the apoptotic cells. The disappearance of the nuclear pore marker accompanied apoptotic progression as judged by the degree of chromatin condensation and DNA fragmentation as analyzed by DNA staining and TUNEL assay, respectively. In contrast, the intensity of the nuclear rim fluorescence was unaffected in necrotic cells displaying an abnormal morphology with tilted nuclei. Thus, it was possible to distinguish between apoptotic and necrotic development in living cells using fluorescence microscopy. This cell line provides a fast and convenient model for screening suspected toxic xenobiotics.     

Quantitative image based apoptotic index measurement using multispectral imaging flow cytometry: a comparison with standard photometric methods

Morphological characterization by microscopy remains the gold standard for accurately identifying apoptotic cells using characteristics such as nuclear condensation, nuclear fragmentation, and membrane blebbing. However, quantitative measurement of apoptotic morphology using microscopy can be time consuming and can lack objectivity and reproducibility, making it difficult to identify subtle changes in large populations. Thus the apoptotic index of a sample is commonly measured by flow cytometry using a variety of fluorescence intensity based (photometric) assays which target hallmarks of apoptosis with secondary markers such as the TUNEL (Terminal Deoxynucleotide Transferase dUTP Nick End Labeling) assay for detection of DNA fragmentation, the Annexin V assay for surface phosphatidylserine (PS) exposure, and fluorogenic caspase substrates to detect caspase activation. Here a novel method is presented for accurate quantitation of apoptosis based on nuclear condensation, nuclear fragmentation, and membrane blebbing using automated image analysis on large numbers of images collected in flow by the ImageStream multispectral imaging cytometer. Additionally the measurement of nuclear fragmentation correlates with the secondary methods of detection of apoptosis over time, indicating that it is also an early marker for apoptosis. False-positive and false-negative events associated with each photometric flow cytometry based method are quantitated and can be automatically removed/included where appropriate. Acquisition of multi-spectral imagery on large numbers of cells couples the quantitative advantage of flow cytometry with the accuracy of morphology-based algorithms allowing more complete and robust analysis of apoptosis.     

Monoclonal Antibody to Single-Stranded DNA Is a Specific and Sensitive Cellular Marker of Apoptosis

The most widely used histochemical marker of apoptosis (in situend labeling, TUNEL) detects both apoptotic and necrotic cells and evaluates only late stages of apoptosis. Hence, a specific and sensitive cellular marker of apoptosis is needed to determine the role of apoptotic death in biology and pathology. The present study describes a novel immunohistochemical procedure for the staining of apoptotic cells using a monoclonal antibody (MAb) to single-stranded DNA. This MAb stained all cells with the morphology typical of apoptosis in etoposide-treated HL-60, MOLT-4, and R9 cell cultures, in which apoptosis was accompanied by high, moderate, and low levels of internucleosomal DNA fragmentation, respectively. TUNEL stained all apoptotic cells in HL-60 cultures, nearly 60% of apoptotic cells in MOLT-4 cultures, and only 14% of apoptotic cells in R9 cultures. Apoptotic R9 cells, which progressed into secondary necrosis, retained MAb staining and became TUNEL-positive. Necrotic cells in MOLT-4 cultures treated with sodium azide were stained by TUNEL, but were negative for MAb staining. All floating cells at a late stage of apoptosis in MDA-MB-468 cultures treated with cisplatin were stained by both MAb and TUNEL. However, among adherent cells in the early stages of apoptosis, MAb stained nearly 20 times more cells than TUNEL. In histological sections of human tumor xenografts, MAb detected clusters of apoptotic cells in viable tumor tissue, but did not stain cells in areas of central ischemic necrosis. In contrast, TUNEL stained nuclei in necrotic areas. Thus, MAb to single-stranded DNA is a specific and sensitive cellular marker of apoptosis, which differentiates between apoptosis and necrosis and detects cells in the early stages of apoptosis.     

Analysis of apoptosis by cytometry using TUNEL assay

Activation of endonucleases that cleave chromosomal DNA preferentially at internucleosomal sections is a hallmark of apoptosis. DNA fragmentation revealed by the presence of a multitude of DNA strand breaks, therefore, is considered to be the gold standard for identification apoptotic cells. Several variants of the methodology that is based on fluorochrome-labeling of 3'-OH termini of DNA strand breaks in situ with the use of exogenous terminal deoxynucleotidyl transferase (TdT), commonly defined as the TUNEL assay, have been developed by us. This Chapter describes the variant based on strand breaks labeling with Br-dUTP that is subsequently detected immunocytochemically with Br-dUAb. Compared with other TUNEL variants the Br-dU-labeling assay offers the greatest sensitivity in detecting DNA breaks. Described also are modifications of the protocol that allow one to use other than Br-dUTP fluorochrome-tagged deoxynucleotides to label DNA breaks. Concurrent staining of DNA with propidium or 4',6-diamidino-2-phenylindole (DAPI) and multiparameter analysis of cells by flow- or laser scanning cytometry enables one to correlate induction of apoptosis with the cell cycle phase.     

Apoptosis and Desquamation of Urothelial Cells in Tissue Remodeling During Rat Postnatal Development

Postnatal rat urothelium was studied from day 0 to day 14, when intense cell loss as part of tissue remodeling was expected. The morphological and biochemical characteristics of urothelial cells in the tissue and released cells were investigated by light and electron microscopy, by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) assay, by annexin V/propidium iodide assay, and by immunofluorescent detection of active caspases and tight-junction protein occludin. Intense apoptosis and massive desquamation were detected between postnatal days 7 and 10. During this period, active caspases and TUNEL-positive cells were found in the urothelium. Disassembled cell–cell junctions were detected between cells. The majority of desquamated cells expressed no apoptotic cell morphology, but were active caspase positive and TUNEL positive. Ann+/PI− apoptotic bodies and desquamated Ann+/PI+ cells were detected in the lumen. These results indicate that apoptosis and desquamation participate in urothelial cell loss in the rat early postnatal period, indispensable for fast urothelial remodeling during development. (J Histochem Cytochem 57:721–730, 2009)     

Flow cytometry analysis of single-strand DNA damage in neuroblastoma cell lines using the F7-26 monoclonal antibody.

The F7-26 monoclonal antibody (Mab) has been reported to be specific for single-strand DNA damage (ssDNA) and to also identify cells in apoptosis. We carriedout studies to determine if F7-26 binding measured by flow cytometry was able to specifically identify exogenous ssDNA as opposed to DNA damage from apoptosis. Neuroblastoma cells were treated with melphalan (L-PAM), fenretinide, 4-hydroperoxycyclophosphamide (4-HC)+/-pan-caspase inhibitor BOC-d-fmk, topotecan or with 10Gy gamma radiation+/-hydrogen peroxide (H2O2) and fixed immediately postradiation. Cytotoxicity was measured by DIMSCAN digital imaging fluorescence assay. The degree of ssDNA damage was analyzed by flow cytometry using Mab F7-26, with DNA visualized by propidium iodide counterstaining. Flow cytometry was used to measure apoptosis detected by terminal deoxynucleotidyltransferase (TUNEL) assay and reactive oxygen species (ROS) by carboxy-dichlorofluorescein diacetate. Irradiated and immediately fixed neuroblastoma cells showed increased ssDNA, but not apoptosis by TUNEL (TUNEL-negative). 4-HC or L-PAM+/-BOC-d-fmk increased ssDNA (F7-26-positive), but BOC-d-fmk prevented TUNEL staining. Fenretinide increased apoptosis by TUNEL but not ssDNA damage detected with F7-26. Enhanced ssDNA in neuroblastoma cells treated with radiation+H2O2 was associated with increased ROS. Topotecan increased both ssDNA and cytotoxicity in 4-HC-treated cells. These data demonstrate that Mab F7-26 recognized ssDNA due to exogenous DNA damage, rather than apoptosis. This assay should be useful to characterize the mechanism of action of antineoplastic drugs.     

Critical evaluation of techniques to detect and measure cell death--study in a model of UV radiation of the leukaemic cell line HL60.

The reliability of eight distinct methods (Giemsa staining, trypan blue exclusion, acridine orange/ethidium bromide (AO/EB) double staining for fluorescence microscopy and flow cytometry, propidium iodide (PI) staining, annexin V assay, TUNEL assay and DNA ladder) for detection and quantification of cell death (apoptosis and necrosis) was evaluated and compared. Each of these methods detects different morphological or biochemical features of these two processes. The comparative analysis of the 8 techniques revealed that AO/EB (read in fluorescence microscopy) provides a reliable method to measure cells in different compartments (or pathways) of cell death though it is very time consuming. PI staining and TUNEL assay were also sensitive in detecting very early signs of apoptosis, but do not allow precise quantification of apoptotic cells. These three methods were concordant in relation to induction of apoptosis and necrosis in HL60 cells with the various UV irradiation time periods tested. Both AO/EB (read by flow cytometry) and annexin V-FITC/PI failed to detect the same number of early apoptotic cells as the other three methods. Trypan blue is valueless for this purpose. Giemsa and DNA ladder might be useful as confirmatory tests in some situations.