Supravital exposure to propidium iodide identifies apoptosis on adherent cells

BACKGROUND: Several studies indicate that plasma membrane changes during apoptosis are a general phenomenon. Among the flow cytometric methods to measure apoptosis, the Annexin V assay that detects the membrane exposure of phosphatidylserine (PS) is one of the most commonly used. However, the various treatments used for the detachment of adherent cells generally interfere with the binding of Annexin V to membrane PS, making apoptosis measurement a technical problem. Materials and Methods Apoptosis of different cell lines was investigated by fluorescence microscopy and multiple flow assays designed to assess loss of membrane integrity, translocation of PS, DNA fragmentation, and light scatter changes. Results and Conclusions We show that supravital propidium iodide (PI) assay stains adherent apoptotic cells, allowing flow cytometric quantification. Moreover, supravital exposure to PI without prior permeabilization identifies apoptotic cells as well as Annexin V and permits the simultaneous surface staining by FITC- and PE-conjugated monoclonal antibodies. As in the case of necrotic or permeabilized cells, fluorescence microscopy has revealed that PI staining of apoptotic cells is localized in the nucleus. This suggests that the binding of PI to the DNA/RNA structures is stable enough to withstand the trypsinization and/or washing procedures necessary to detach adherent cells.     

Discrimination between primary necrosis and apoptosis by necrostatin-1 in Annexin V-positive/propidium iodide-negative cells

Apoptotic cell death eventually results in secondary necrotic cell death, whereas caspase-independent primary necrotic cell death has been reported and its mechanism involving RIP1 and RIP3 has been recently elucidated. Dual staining with fluorescent Annexin V and propidium iodide (PI) has been used to discriminate apoptotic and necrotic cell death, in which Annexin V-positive/PI-negative staining is regarded as apoptosis and PI-positive staining as necrosis. Here we demonstrate that primary necrotic cells unexpectedly show Annexin V-positive/PI-negative staining before they become PI-positive, and that primary necrotic and apoptotic Annexin V-positive/PI-negative cells can be discriminated by necrostatin-1, an inhibitor of primary necrosis by inhibition of RIP1.     

Simple discrimination of sub-cycling cells by propidium iodide flow cytometric assay in Jurkat cell samples with extensive DNA fragmentation

Human leukemia Jurkat T cells were analyzed for apoptosis and cell cycle by flow cytometry, using the Annexin V/propidium iodide (PI) standard assay, and a simple PI staining in Triton X-100/digitonin-enriched PI/RNase buffer, respectively. Cells treated with doxorubicin or menadione displayed a very strong correlation between the apoptotic cell fraction measured by the Annexin V/PI assay, and the weight of a secondary cell population that emerged on the forward scatter (FS)/PI plot, as well as on the side scatter (SS)/PI and FL1/PI plots generated from parallel cell cycle recordings. In both cases, the Pearson correlation coefficients were >0.99. In cell cycle determinations, PI fluorescence was detected on FL3 (620/30 nm), and control samples exhibited the expected linear dependence of FL3 on FL1 (525/40 nm) signals. However, increasing doses of doxorubicin or menadione generated a growing subpopulation of cells displaying a definite right-shift on the FS/FL3, SS/FL3 and FL1/FL3 plots, as well as decreased PI fluorescence, indicative of ongoing fragmentation and loss of nuclear DNA. By gating on these events, the resulting fraction of presumably sub-cycling cells (i.e. cells with cleaved DNA, counting sub-G₀/G₁, sub-S and sub-G₂/M cells altogether) was closely similar to the apoptotic rate assessed by Annexin V/PI labeling. Taken together, these findings suggest a possible way to recognize the entire population of cells undergoing apoptotic DNA cleavage and simultaneously determine the cell cycle distribution of non-apoptotic cells in PI-labeled cell samples with various degrees of DNA fragmentation, using a simple and reproducible multiparametric analysis of flow cytometric recordings.     

Flow cytometric analysis of apoptotic subpopulations with a combination of annexin V-FITC, propidium iodide, and SYTO 17

BACKGROUND: We have previously characterized apoptotic cell death induced in a follicular lymphoma cell line, HF-1, after triggering via the B-cell receptor (BCR) or treatment with Ca(2+) Ionophore A23187. We analyzed the kinetics of apoptosis induced by these two treatments, as two alternative models of classical apoptosis, by flow cytometry using a novel combination of cytofluorometric stains. METHODS: Cells were stained with a combination of Annexin V-FITC, propidium iodide (PI), and SYTO 17 and analyzed by a two-laser flow cytometry system using 488-nm argon and 633-nm HeNe air-cooled lasers. RESULTS: In both apoptotic models, the first apoptotic cells were detected by SYTO 17 staining. The alteration in SYTO 17 staining intensity was followed by an increased uptake of PI. Finally, the apoptotic cells were labeled with Annexin V in BCR-induced apoptosis. On the contrary, on treatment with Ca(2+) Ionophore A23187, cells became positive for Annexin V earlier than for PI. CONCLUSIONS: The novel cytofluorometric dye, SYTO 17, discriminates apoptotic alterations before Annexin V and PI. PI also discriminates apoptotic alterations before the loss of plasma membrane asymmetry by BCR but not by Ca(2+) Ionophore A23187-induced apoptosis. Finally, the combination of these three cytofluorometric dyes allows effective detection of apoptotic subpopulations and ordering of apoptotic events by flow cytometry.     

Sperm apoptosis in fresh and cryopreserved bull semen detected by flow cytometry and its relationship with fertility.

The present study was conducted to detect sperm apoptosis in fresh and frozen semen and to determine its relationship with bull fertility. Three ejaculates were collected from five breeding bulls with different fertility levels and were cryopreserved using standard methods. Two flow cytometric methods were employed to measure apoptosis: an assay for phosphatidylserine (PS) translocation across the plasma membranes using fluorescein-labeled Annexin V and propidium iodide (PI), and an assay for nicked DNA using bromodeoxyuridine (BrdU), terminal deoxynucleotidyl transferase, and fluorescein-labeled anti-BrdU monoclonal antibody. Both assays showed that fresh sperm contained 10%-20% apoptotic sperm. Significant differences in the percentage of apoptotic sperm were observed among the bulls. Cryopreservation induced translocation of PS to the outer leaflet of the plasma membrane and caused most of the necrotic cells in fresh sperm to disintegrate. Bull fertility was significantly related to the percentage of necrotic or viable sperm in fresh semen as detected by the Annexin V/PI assay, to the number of apoptotic sperm in fresh semen as detected by the TUNEL assay, and to the level of chromatin or DNA condensation as detected by PI staining. The present study suggests that the presence of apoptotic spermatozoa in fresh semen could be one of the reasons for poor fertility in breeding bulls.     

Analysis of radiation-induced apoptosis in human lymphocytes: flow cytometry using Annexin V and propidium iodide versus the neutral comet assay

BACKGROUND: The neutral comet assay was devised to measure double-stranded DNA breaks, but it has also been used to measure apoptosis based on its characteristic DNA fragmentation patterns. There is still uncertainty about the reliability of this method. By comparing the comet assay with a flow cytometry method that uses Annexin V binding to apoptotic cells, we have provided further evidence for evaluating the usefulness of the comet assay for detecting apoptosis. METHODS: Apoptosis was induced in human peripheral blood mononuclear cells (PBMC) by ionizing radiation and measured using the comet assay and a flow cytometry method that measures Annexin V and propidium iodide (PI) staining. RESULTS: The Annexin V flow cytometry assay distinguished among early apoptosis, late apoptosis, and an apoptotic or necrotic phase in which the cells were labeled with both Annexin V and PI. The comet assay detected only the latter two phases of apoptosis. CONCLUSIONS: The comet assay is a useful tool for measuring the late stages of apoptosis whereas the Annexin V assay measures higher amounts of apoptosis because it can detect cells in an earlier stage of the apoptotic pathway.     

Quinidine and procainamide inhibit murine macrophage uptake of apoptotic and necrotic cells: A novel contributing mechanism of drug-induced-lupus

A number of mechanisms have been proposed to explain the etiology of drug-induced lupus (DIL) but the effect of apoptotic and necrotic cell handling has not been previously examined.Objective. To evaluate the effect of quinidine and procainamide at therapeutic range concentrations, on the uptake of apoptotic and necrotic thymocytes by murine peritoneal macrophages and on macrophage survival, as a novel mechanism for DIL.Methods. Thymocytes were stained and induced to undergo apoptosis by serum withdrawal. Apoptosis was evaluated using annexin V and propidum iodide (PI) and PI staining. Necrosis was induced by heating. Peritoneal macrophages were treated with quinidine or procainamide at a range of therapeutic concentrations and incubated with stained apoptotic and necrotic thymocytes. Apoptotic and necrotic cell uptake was evaluated by flow cytometry using double staining of thymocytes and macrophages and by confocal microscopy. Green fluorescent latex beads were used as controls for phagocytosis.Results. Significantly decreased uptake of apoptotic and necrotic cells was seen in the presence of quinidine and procainamide. The documented effect was mainly on the number of apoptotic/necrotic cells per macrophage. Uptake of fluorescent latex beads offered to resident macrophages was not significantly affected by quinidine or procainamide. No pro-apoptotic effect of quinidine or procainamide on macrophages was seen.Conclusion. Quinidine and procainamide at therapeutic range concentrations specifically inhibit clearance of apoptotic and necrotic cells by peritoneal macrophages. Altered handling of apoptotic and necrotic cells may represent a contributing mechanism for DIL.     

Anti-Candida activity of two-peptide bacteriocins,plantaricins (Pln E/F and J/K) and their mode of action

The fungicidal effect of plantaricin peptides PlnE, -F, -J, and -K was studied against pathogenic yeast, Candida albicans. Dose-dependent inhibitory effect was observed by drop in cell viability, further demonstrated by measuring the fluorescence intensity of cells by exposing them to 5, (6)-carboxyfluorescein diacetate (CFDA). Live/dead staining by CFDA and propidium iodide (PI) also suggested the viability loss response. Also, the PI uptake by treated cells suggested the membrane damage. PlnJ was identified as most inhibitory among different plantaricins tested. PlnJ not only induced membrane potential dissipation but also resulted in the release of K⁺. In addition, enhanced production of reactive oxygen species (ROS) was also observed by fluorometry using 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA). Dual staining with Hoechst stain and PI depicted both early apoptotic and necrotic cells in the treated population. Terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) positive staining further confirmed the ROS-mediated apoptosis. Scanning electron microscopy and transmission electron microscopy also revealed characteristic apoptotic features such as appearance of blebs, indentations, and wrinkling of the cell wall, discontinuity of cell membrane, undefined and damaged nuclei, and shrinkage of protoplasm. Taken together the results suggest that Pln-treatment initiate the apoptosis cell death which may lead to necrosis due to toxicity of the plantaricin peptides.     

Pyrrolidine dithiocarbamate (PDTC) blocks apoptosis and promotes ionizing radiation-induced necrosis of freshly-isolated normal mouse spleen cells

Ionizing radiation (IR) is a pro-oxidant that kills cells by both apoptotic and necrotic mechanisms. Pyrrolidine dithiocarbamate (PDTC) is a thiol-containing compound that may act either as a pro- or anti-oxidant depending on the experimental conditions. This study was designed to determine whether PDTC would reduce or enhance IR-induced cell death of freshly-isolated normal mouse B6/129 spleen cells (NMSC). We determined the effect of increasing doses of IR, PDTC alone and PDTC followed by IR on the viability of NMSC. Annexin V and propidium iodide (Annexin V/PI) staining demonstrated a dose and time-dependent relationship in which PDTC enhanced the percentage of IR-induced apoptotic/necrotic NMSC. Trypan blue dye inclusion confirmed that a loss of membrane integrity was occurring 1 h after incubation with PDTC plus IR. Reduction in the glutathione (GSH)/glutathione disulfide (GSSG) ratio and GSH demonstrated that both IR (8.5 Gy) and PDTC acted as pro-oxidants, but their mechanisms of action differed: In contrast to IR, which promoted p53 activation and caspase 3/7-mediated apoptosis, PDTC inhibited IR-induced p53 and caspase 3/7 activity. However, PDTC increased H₂O₂ formation and necrosis, resulting in an overall increase in IR-induced cell death. Catalase prevented the PDTC-induced increase in IR cytotoxicity implicating the generation of H₂O₂ as a major factor in this mechanism. These results demonstrate that in NMSC PDTC acts as pro-oxidant and enhances IR-induced cell cytotoxicity by increasing H₂O₂formation and thiol oxidation. As such, they strongly suggest that the use of PDTC as an adjunct to reduce radiation toxicity should be avoided.     

Matrin 3 as a key regulator of endothelial cell survival

Matrin 3 is an integral component of nuclear matrix architecture that has been implicated in interacting with other nuclear proteins and thus modulating the activity of proximal promoters. In this study, we evaluated the contribution of this protein to proliferation of endothelial cells. To selectively modulate matrin 3 expression, we used siRNA oligonucleotides and transfection of cells with a pEGFP-N1-Mtr3. Our data indicate that downregulation of matrin 3 is responsible for reduced proliferation and leads to necrosis of endothelial cells. This conclusion is supported by observations that reducing matrin 3 expression results in (a) producing signs of necrosis detected by PI staining, LDH release, and scatter parameters in flow cytometry, (b) affecting cell cycle progression. It does not cause (c) membrane asymmetry of cells as indicated by lack of Annexin V binding as well as (d) activation of caspase 3 and cleavage of PARP. We conclude that matrin 3 plays a significant role in controlling cell growth and proliferation, probably via formation of complexes with nuclear proteins that modulate pro- and antiapoptotic signaling pathways. Thus, degradation of matrin 3 may be a switching event that induces a shift from apoptotic to necrotic death of cells.     

Membrane dielectric changes indicate induced apoptosis in HL-60 cells more sensitively than surface phosphatidylserine expression or DNA fragmentation

The specific membrane capacitance and conductivity of mammalian cells, which reflect their surface morphological complexities and membrane barrier functions, respectively, have been shown to respond to cell physiologic and pathologic changes. Here, the effects of induced apoptosis on these membrane properties of cultured human promyelocytic HL-60 cells are reported. Changes in membrane capacitance and conductivity were deduced from measurements of cellular dielectrophoretic crossover frequencies following treatment with genistein (GEN). The apparent specific cell membrane capacitance of HL-60 cells fell from an initial value of 17.6±0.9 to 9.1±0.5 mF/m² 4 h after treatment. Changes began within minutes of treatment and preceded both the externalization of phosphatidylserine (PS), as gauged by the Annexin V assay, and the appearance of a sub-G1 cell subpopulation, as determined through ethidium bromide staining of DNA. Treatment by the broad spectrum caspase inhibitor N-benzyloxycarbony-Val-Ala-Asp(O-methyl)-fluoromethyketone (zVAD-fmk) did not prevent these early cell membrane dielectric responses, suggesting that the caspase system was not involved. Although membrane conductivity did not alter during the first 4 h of GEN treatment, it rose significantly and progressively thereafter. Finally, as the barrier function failed and the cells became necrotic, it increased by many orders of magnitude. The effective membrane capacitance and conductivity findings serve to focus attention on the membrane as a site for early participation in apoptosis. In conjunction with our prior reports of the use of dielectric methods for cell manipulation and separation, these results demonstrate that dielectrophoretic technologies should be applicable to the rapid detection, separation, and quantification of normal, apoptotic, and necrotic cells from cell mixtures.     

Induction of yeast apoptosis by an antimicrobial peptide, Papiliocin

Papiliocin is a 37-residue peptide isolated from the swallowtail butterfly Papilio xuthus. In this study, we found that Papiliocin induced the accumulation of reactive oxygen species (ROS) and hydroxyl radicals known to be important regulators of apoptosis in Candida albicans. To examine the relationship between the accumulation of ROS and the induction of apoptosis, we investigated the apoptotic effects of Papiliocin using apoptotic markers. Cells treated with Papiliocin showed a series of cellular changes normally seen in cells undergoing apoptosis: plasma membrane translocation of phosphatidylserine from the inner to the outer membrane leaflet, measured by Annexin V staining, dissipation of the mitochondrial membrane potential, observed by DiOC₆(3) staining; and the presence of active metacaspases, measured using the CaspACE FITC-VAD-FMK, as early apoptotic events. In addition, DNA condensation and fragmentation, which is important marker of late stage apoptosis, was seen by DAPI and TUNEL assay. Therefore, these results suggest that Papiliocin leads to apoptosis in C. albicans via ROS accumulation.     

Annexin V staining due to loss of membrane asymmetry can be reversible and precede commitment to apoptotic death.

Signal-induced apoptosis is a normal phenomenon in which cells respond to changes in their environment through a cascade of intracellular biochemical changes culminating in cell death. However, it is not clear at what point in this process the cell becomes committed to die. An early biochemical change characteristic of cells undergoing apoptosis is the loss of plasma membrane asymmetry, such that high levels of phosphatidylserine become exposed on the outside cell surface. These cells can be recognized by staining with Annexin V, which binds to phosphatidylserine with high affinity. To investigate the mechanisms controlling signal-induced apoptosis we have examined the response of a B cell lymphoma to crosslinking of the membrane immunoglobulin (mIg) receptor. We have found that many of the cells that stain positive for Annexin V are viable and can resume growth and reestablish phospholipid asymmetry once the signal is removed. These results indicate that Annexin V staining, and thus loss of membrane asymmetry, precedes commitment to apoptotic death in this system.     

Plasma membrane phospholipid asymmetry precedes DNA fragmentation in different apoptotic cell models

 Biochemical alterations occurring in many cell types during apoptosis include the loss of plasma membrane phospholipid asymmetry and nuclear DNA fragmentation. Annexin V staining detects phosphatidylserine translocation into the outer plasma membrane layer occurring during cell death, while the in situ tailing (IST or TUNEL) reaction labels the DNA strand breaks typical of apoptosis. To compare the time course of these processes we investigated methylprednisolone-induced apoptosis of rat thymocytes, topoisomerase inhibitor-induced apoptosis in the human histiocytic lymphoma cell line U937, and serum deprivation-induced apoptosis in the rat pheochromocytoma cell line, PC12. At all time points, FACS analysis and quantitative fluorescence light microscopy showed a higher proportion of annexin V-positive than IST-positive cells, with significantly different time courses in the apoptotic cell models investigated (Anova test). Results were confirmed by confocal microscopy. Our data indicate that the exposure of phosphatidylserine, a potential phagocyte recognition signal on the cell surface of apoptotic cells in vivo, precedes DNA strand breaks during apoptosis in different cell types. Accepted: 29 June 1998     

Assessment of fresh and frozen-thawed boar semen using an Annexin-V assay: a new method of evaluating sperm membrane integrity

Detection of early changes in the sperm plasma membrane during cryopreservation is of utmost importance when designing freezing protocols. The present study evaluated the ability of an Annexin-V binding assay to detect early changes in sperm membrane integrity using flow cytometry (FC) in two different portions of the boar ejaculate, in cryopreserved semen. Using a split sample design, sperm motility was evaluated in fresh (controls) and frozen-thawed (FT) samples, both subjectively and by means of a computer-assisted motility assessment (CASA) system, while membrane integrity was assessed using Annexin-V (A) and propidium iodide (PI) staining in spermatozoa derived from the first sperm-rich fraction (Portion I) or the remaining ejaculate (Portion II). The A/PI technique revealed four sperm subpopulations, two PI negative (either A- (alive) or A+ (apoptotic)); and two PI positive (dead cells), either A+ (dead, late apoptotic or early necrotic cells) or A- (dead, late necrotic cells). Significant differences were found between the two portions of the ejaculate in the fresh (control) and FT samples. In the fresh controls, significantly more live, nonapoptotic spermatozoa (A-/PI-) were present in Portion I than in Portion II (P<0.001). Although apoptotic spermatozoa were detected in both semen portions, the frequency of live, early apoptotic (A+/PI-) cells was significantly lower in Portion I than in Portion II (P<0.001). Irrespective of the ejaculate portion considered, freezing and thawing significantly decreased the mean percentages of live spermatozoa (P<0.01), and dramatically increased the percentages of apoptotic or early necrotic cells (P<0.01), but not of early apoptotic cells (N.S.). The latter finding might suggest that apoptotic changes due to cryopreservation using the procedures applied in this trial are transient and lead to cell death. In conclusion, the Annexin-V binding assay was able to detect deleterious changes in the sperm plasma membrane at an earlier point than PI staining, thus representing a novel approach to investigating membrane integrity in this species. The finding that fewer spermatozoa in Portion I of the ejaculate showed early apoptosis post-freezing, suggests boar spermatozoa in this portion of the seminal plasma are less sensitive to the stress induced by cryopreservation.     

Multiparametric study of atresia in ewe antral follicles: histology, flow cytometry, internucleosomal DNA fragmentation, and lysosomal enzyme activities in granulosa cells and follicular fluid

The differential quantitative participation of apoptosis and necrosis in ewe antral follicles of two different sizes, separated in four stages of atresia using macroscopic, histologic, and esteroid quantification methods was assessed. Annexin V binding and propidium iodide (PI) uptake was used to detect healthy live cells (Annexin V negative/PI negative), early apoptotic cells (Annexin V+/PI-), and necrotic or late apoptotic cells (PI+). Additionally we used internucleosomal DNA fragmentation as a quantitative estimate of apoptosis. Presence and distribution of lysosomal enzymes in follicular fluid and granulosa cells was used as a measure of necrotic cell death. DNA flow cytometry and gel electrophoresis were positively correlated with the progression of atresia, small atretic follicles tend to have higher percentages of internucleosomal cleaved DNA than follicles >6 mm. Annexin/PI binding also indicates that apoptosis and necrosis increase with atresia progression, generally apoptosis outweighs necrosis in small follicles. Acid phosphatase and glucosaminidase in follicular fluid of 3-6 mm follicles showed no significant modifications between healthy and initially atretic follicles, and only a small, but significant increase in activity in advancedly atretic follicles. On the contrary, lysosomal enzyme activity in follicles >6 mm showed positive correlation between atresia stages and the activities of acid phosphatase and glucosaminidase in follicular fluid. A similar size-differential behavior was found in free or membrane-bound lysosomal enzyme activity of granulosa cells. Necrosis, but principally apoptosis, were present during all stages of follicular maturation indicating that growth and maturation of ovarian follicles involves a continuous renewal of granulosa cells, regulated by apoptosis. Mechanisms regulating this equilibrium may participate in the final destiny, whether ovulation or atresia of ovarian follicles.     

Antiproliferation and apoptosis of human tumor cell lines by a lectin (AMML) of Astragalus mongholicus

A lectin (AMML) from the roots of Astragalus mongholicus was extracted and purified by affinity chromatographic technique. Human cervical carcinoma cell line (HeLa), human osteoblast-like cell line (MG63) and human leukemia cell line (K562) were used to check the effects of AMML on cell proliferation, apoptosis and cell cycle. Maximum growth inhibition (92%) was observed with HeLa cells, followed by K562 cells (84%) and MG63 (48%) cells. Morphological observation showed that AMML-treated HeLa cells displayed outstanding apoptosis characteristics, such as nuclear fragmentation and appearance of membrane-enclosed apoptotic bodies. The apoptosis of HeLa cells was confirmed by flow cytometry using Annexin V/FITC and propidium iodide (PI) staining technique. For the first time we also report a significant cell cycle arrest at S phase of HeLa cells by AMML. Therefore, the present investigation may lead to the possible therapeutic use of Astragalus mongholicus lectin.     

Phosphatidylserine plasma membrane asymmetry in vivo: a pancellular phenomenon which alters during apoptosis

The distribution of phospholipids across the two leaflets of the plasma membrane is important for many cellular processes including phagocytosis and hemostasis. In the present study we investigated the in vivo plasma membrane distribution of the aminophospholipid phosphatidylserine in mouse embryos with a novel technique employing Annexin V, a Ca2+ dependent phosphatidylserine binding protein, conjugated to fluorescein isothiocyanate and biotin. Annexin V directly applied to cryostat sections labeled the plasma membrane of all cells at the interface. In contrast, Annexin V injected intracardially into viable mouse embryos labeled almost exclusively apoptotic cells. These apoptotic cells were visible in all tissues and derived from all germ layers. Our experiments demonstrate that phosphatidylserine is asymmetrically distributed between the two leaflets of the plasma membrane in virtually all cell types in vivo and that this asymmetry is lost early during apoptosis.     

Apoptosis phenomenon in the schistosomulum and adult worm life cycle stages of Schistosoma japonicum

Apoptosis is an important aspect of a number of biological processes, from embryogenesis to the stress–injury response. It plays a central role in balancing cell proliferation and tissue remodeling activity in many organisms. In the present study, apoptosis in 14 days post infection schistosomula was evaluated using TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assays and DAPI staining. Additionally, flow cytometry using the Annexin V-FITC/propidium iodide (PI) (Annexin V/PI) assay confirmed the percentage of early apoptotic, late apoptotic, and necrotic cells in 14 and 23 days post infection worms. Conserved Domain Database (CDD) BLAST analysis and alignment analysis of known schistosome proteins demonstrated the feasibility of detecting the activity of caspase-3 and -7 using the caspase-3/7 Glo analysis assay. Analysis of caspase-3 and -7 activities in schistosome demonstrated that both caspases were active in each developmental stage of Schistosoma japonicum, but was highest in the 14 days post infection schistosomula. Additionally, the caspase peptide inhibitor (Z-VAD-FMK) inhibited the caspase-3/7 activity at all developmental stages examined. Therefore, we hypothesized that two main signaling pathways are involved in apoptosis in S. japonicum, the caspase cascade and the mitochondrial-initiated pathway. We have constructed a model of these two pathways, including how they may interact and their biological outcomes. qRT-PCR analyses of the gene expression profiles of apoptosis-related genes supported our hypothesis of the relationship between the apoptotic pathway and parasite development. The data presented here demonstrates that apoptosis is an important biological process for the survival and development of the schistosome, and identifies potential novel therapeutic targets.