scholarly journals Caspofungin Kills Candida albicans by Causing both Cellular Apoptosis and Necrosis

2012 ◽  
Vol 57 (1) ◽  
pp. 326-332 ◽  
Author(s):  
Binghua Hao ◽  
Shaoji Cheng ◽  
Cornelius J. Clancy ◽  
M. Hong Nguyen
Keyword(s):  
Cell Death ◽  
Candida Albicans ◽  
Programmed Cell Death ◽  
Membrane Integrity ◽  
Annexin V ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Dapi Staining ◽  
Content Type ◽  
Glucan Synthesis ◽  
Apoptosis And Necrosis

ABSTRACTCaspofungin exerts candidacidal activity by inhibiting cell wall (1,3)-β-d-glucan synthesis. We investigated the physiologic mechanisms of caspofungin-inducedCandida albicanscell death. Apoptosis (programmed cell death) and necrosis were studied afterC. albicansSC5314 cells were exposed to caspofungin at 0.06, 0.125, and 0.5 μg/ml (0.5×, 1×, and 4× the MIC, respectively) for 3 h. Caspofungin at 0.125 and 0.5 μg/ml reduced cellular viability by >50%, as measured by colony counts and methylene blue exclusion. Apoptosis and necrosis were demonstrated by annexin V and propidium iodide staining for phosphatidylserine externalization and loss of membrane integrity, respectively. At all concentrations of caspofungin, 20 to 25% and 5 to 7% ofC. albicanscells exhibited early apoptosis and late apoptosis/necrosis, respectively (Pvalue was not significant [NS]). Necrosis, on the other hand, was significantly greater at 0.125 (43%) and 0.5 (48%) μg/ml than at 0.06 μg/ml (26%) (Pvalues of 0.003 and 0.003, respectively). The induction of apoptosis at concentrations less than or equal to the MIC was corroborated by dihydrorhodamine 123 (DHR-123) and dihydroethidium (DHE) staining (reactive oxygen species production), JC-1 staining (mitochondrial membrane potential dissipation), and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining (DNA damage and nuclear fragmentation). Moreover, electron microscopy of cells exposed to 0.125 μg/ml of caspofungin showed hallmark apoptotic features like chromatin margination and condensation and nuclear blebs. Apoptosis was associated with metacaspase 1 activation, as demonstrated by D2R staining. Caspofungin exerts activity againstC. albicansby directly killing cells (resulting in necrosis) and causing others to undergo programmed cell death (apoptosis). Apoptosis is initiated at subinhibitory concentrations, suggesting that strategies to target this process may augment the benefits of antifungal agents.

scholarly journals The Als3 Cell Wall Adhesin Plays a Critical Role in Human Serum Amyloid A1-Induced Cell Death and Aggregation in Candida albicans

2020 ◽  
Vol 64 (6) ◽  
Author(s):  
Jiao Gong ◽  
Jian Bing ◽  
Guobo Guan ◽  
Clarissa J. Nobile ◽  
Guanghua Huang
Keyword(s):  
Cell Death ◽  
Cell Wall ◽  
Candida Albicans ◽  
Critical Role ◽  
Membrane Integrity ◽  
Cell Aggregation ◽  
Serum Amyloid ◽  
Mouse Serum ◽  
Content Type ◽  
Serum Amyloid A1

ABSTRACT Antimicrobial peptides and proteins play critical roles in the host defense against invading pathogens. We recently discovered that recombinantly expressed human and mouse serum amyloid A1 (rhSAA1 and rmSAA1, respectively) proteins have potent antifungal activities against the major human fungal pathogen Candida albicans. At high concentrations, rhSAA1 disrupts C. albicans membrane integrity and induces rapid fungal cell death. In the present study, we find that rhSAA1 promotes cell aggregation and targets the C. albicans cell wall adhesin Als3. Inactivation of ALS3 in C. albicans leads to a striking decrease in cell aggregation and cell death upon rhSAA1 treatment, suggesting that Als3 plays a critical role in SAA1 sensing. We further demonstrate that deletion of the transcriptional regulators controlling the expression of ALS3, such as AHR1, BCR1, and EFG1, in C. albicans results in similar effects to that of the als3/als3 mutant upon rhSAA1 treatment. Global gene expression profiling indicates that rhSAA1 has a discernible impact on the expression of cell wall- and metabolism-related genes, suggesting that rhSAA1 treatment could lead to a nutrient starvation effect on C. albicans cells.

scholarly journals Analysis of programmed cell death in mouse fetal oocytes

Reproduction ◽  
2007 ◽  
Vol 134 (2) ◽  
pp. 241-252 ◽  
Author(s):  
A M Lobascio ◽  
F G Klinger ◽  
M L Scaldaferri ◽  
D Farini ◽  
M De Felici
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Specific Inhibitor ◽  
Annexin V ◽  
Death Process ◽  
Parp Cleavage ◽  
Tunel Staining ◽  
Calpain Inhibitor ◽  
Dna Ladder ◽  
Caspase 2

We report a short-term culture system that allowsto define novel characteristic of programmed cell death (PCD) in fetal oocytes and to underscore newaspects of this process. Mouse fetal oocytes culturedin conditions allowingmeiotic prophase I progression underwent apoptotic degeneration waves as revealed by TUNEL staining. TEM observations revealed recurrent atypical apoptotic morphologies characterized by the absence of chromatin margination and nuclear fragmentation; oocytes with autophagic and necrotic features were also observed. Further characterization of oocyte death evidenced DNA ladder, Annexin V binding, PARP cleavage, and usually caspase activation (namely caspase-2). In the aim to modulate the oocyte death process, we found that the addition to the culture medium of the pancaspase inhibitors Z-VAD orcaspase-2-specific inhibitor Z-VDVAD resulted in a partial and transient prevention of this process. Oocyte death was significantly reduced by the antioxidant agent NAC and partly prevented by KL and IGF-I growth factors. Finally, oocyte apoptosis was reduced by calpain inhibitor I and increased by rapamycin after prolonged culture.These results support the notion that fetal oocytes undergo degeneration mostly by apoptosis. This process is, however, often morphologically atypical and encompasses other forms of cell death including caspase-independent apoptosis and autophagia. The observation that oocyte death occurs mainly at certain stages of meiosis and can only be attenuated by typical anti-apoptotic treatments favors the notion that it is controlled at least in part by stage-specific oocyte-autonomous meiotic checkpoints and when activated is little amenable to inhibition being the oocyte able to switch back and forth among different death pathways.

Extracellular ATP causes apoptosis and necrosis of cultured mesangial cells via P2Z/P2X7receptors

1998 ◽  
Vol 275 (6) ◽  
pp. F962-F971 ◽  
Author(s):  
Eckhard Schulze-Lohoff ◽  
Christian Hugo ◽  
Sylvia Rost ◽  
Susanne Arnold ◽  
Angela Gruber ◽  
...  
Keyword(s):  
Cell Death ◽  
Dna Fragmentation ◽  
Mesangial Cells ◽  
Lucifer Yellow ◽  
Fold Increase ◽  
Extracellular Atp ◽  
Glomerular Disease ◽  
Terminal Deoxynucleotidyl Transferase ◽  
P53 Expression ◽  
Apoptosis And Necrosis

Mesangial cells undergo cell death both by apoptosis and necrosis during glomerular disease. Since nucleotides are released from injured and destroyed cells in the glomerulus, we examined whether extracellular ATP and its receptors may regulate cell death of cultured mesangial cells. Addition of extracellular ATP (300 μM to 5 mM) to cultured rat mesangial cells for 90 min caused a 5.8-fold increase in DNA fragmentation (terminal deoxynucleotidyl transferase assay) and a 4.2-fold increase in protein levels of the tumor suppressor p53, which is thought to regulate apoptosis. Apoptotic DNA fragmentation was confirmed by the diphenylamine assay and by staining with the DNA-specific fluorochrome Hoechst 33258. The necrotic markers, release of lactate dehydrogenase and uptake of trypan blue, were not positive before 3 h of ATP addition. The effects of ATP on DNA fragmentation and p53 expression were reproduced by the purinergic P2Z/P2X7 receptor agonist, 3′- O-(4-benzoylbenzoyl)-ATP, and inhibited by the P2Z/P2X7 receptor blocker, oxidized ATP. Transcripts encoding the P2Z/P2X7 receptor were expressed by cultured mesangial cells as determined by Northern blot analysis. P2Z/P2X7 receptor-associated pore formation in the plasma membrane was demonstrated by the Lucifer yellow assay. We conclude that activation of P2Z/P2X7 receptors by extracellular ATP causes apoptosis and necrosis of cultured mesangial cells. Activation of purinergic P2Z/P2X7 receptors may play a role in causing death of mesangial cells during glomerular disease.

scholarly journals EXPERIMENTAL MODELING OF APOPTOSIS UNDER CONDITIONS OF STABLE STRONTIUM EXPOSURE

2019 ◽  
Vol 21 (1) ◽  
pp. 137-140
Author(s):  
O. V. Dolgikh ◽  
N. V. Zaitseva ◽  
D. G. Dianova ◽  
A. V. Krivtsov ◽  
K. D. Starkova ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Programmed Cell Death ◽  
Annexin V ◽  
Fold Increase ◽  
Apoptosis Markers ◽  
Stable Strontium ◽  
Cell Death Mechanisms ◽  
Apoptotic Pathways ◽  
And Control

Apoptosis is defined as a highly regulated form of programmed cell death with typical morphological and biochemical features. A variety of factors, including heavy metals, may influence the intensity of programmed cell death. The aim of the work was to simulate apoptosis in an in vitrosystem under the conditions of stable strontium exposure. The children’s population consuming drinking water with high strontium (Sr2+) content (n = 49) was observed. The level of lymphocyte apoptosis was determined with flow cytometry technique, by means of labeled annexin V-FITC conjugate (AnnV-FITC) and propidium iodide (PI) staining. AnnV-FITC+PI- cells were regarded as early apoptotic forms, whereas late apoptotic and/or necrotic cells were AnnV-FITC+PI+. The isolated leukocytes were incubated with Sr2+ at a concentration of 7.0 mg/l, the maximal permitted concentration (MPC) for water of aqueous objects, for 4 hours at 37 ºC. Expression of CD95 and p53 apoptosis markers was performed by flow cytometry using labeled monoclonal antibodies.In vitroexposure to strontium was associated with significantly decreased expression of apoptosisregulating factors, i.e., membrane marker CD95 and intracellular transcription protein p53, 1.56- and 1.68-fold, respectively. Meanwhile, we revealed a significantly (4.68-fold) decreased amounts of AnnV-FITC+PI--cells, as well as a statistically significant (1.35-fold) increase of the AnnV-FITC+PI+-cells. Moreover, the amounts of AnnV-FITC+ PI--lymphocytes in all samples were below the physiological ranges and control values. The number of samples with higher contents of AnnV-FITC+PI+-lymphocyte exceeding the established standards and control values, was 30.8%. Thus, it has been experimentally proven that strontium, at a concentration corresponding to MPC for water objects may significantly inhibit cell death along apoptotic pathways, with switching to necrotic cell death mechanisms, according to phosphatidylserine contents, as detected by annexin V binding test. The data have revealed an ability of strontium to have a significant effect upon the parameters of regulation and maintenance of cellular homeostasis, by influencing the apoptosis intensity, due to shifting a balance towards necrosis and reducing expression of apoptosis-regulating factors. The results of this study may be used in order to identify some marker indexes of immune disorders potentially induced by external influence of strontium upon human health under specific environmental factors.

scholarly journals Flight muscles degenerate by programmed cell death after migration in the wheat aphid, Sitobion avenae

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Honglin Feng ◽  
Xiao Guo ◽  
Hongyan Sun ◽  
Shuai Zhang ◽  
Jinghui Xi ◽  
...  
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Molecular Mechanism ◽  
Differentially Expressed Genes ◽  
Flight Muscle ◽  
Sitobion Avenae ◽  
Differentially Expressed ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Muscle Degeneration ◽  
Flight Muscles

Abstract Objective Previous studies showed that flight muscles degenerate after migration in some aphid species; however, the underlying molecular mechanism remains virtually unknown. In this study, using the wheat aphid, Sitobion avenae, we aim to investigate aphid flight muscle degeneration and the underlying molecular mechanism. Results Sitobion avenae started to differentiate winged or wingless morphs at the second instar, the winged aphids were fully determined at the third instar, and their wings were fully developed at the fourth instar. After migration, the aphid flight muscles degenerated via programmed cell death, which is evidenced by a Terminal deoxynucleotidyl transferase dUTP-biotin nick-end labeling assay. Then, we identified a list of differentially expressed genes before and after tethered flights using differential-display reverse transcription-PCR. One of the differentially expressed genes, ubiquitin-ribosomal S27a, was confirmed using qPCR. Ubiquitin-ribosomal S27a is drastically up regulated following the aphids’ migration and before the flight muscle degeneration. Our data suggested that aphid flight muscles degenerate after migration. During flight muscle degeneration, endogenous proteins may be degraded to reallocate energy for reproduction.

scholarly journals Identification of a Cell Death Pathway in Candida albicans during the Response to Pheromone

2010 ◽  
Vol 9 (11) ◽  
pp. 1690-1701 ◽  
Author(s):  
Kevin Alby ◽  
Dana Schaefer ◽  
Racquel Kim Sherwood ◽  
Stephen K. Jones ◽  
Richard J. Bennett
Keyword(s):  
Cell Death ◽  
Candida Albicans ◽  
Laboratory Strain ◽  
Opportunistic Pathogen ◽  
Cell Types ◽  
Yeast Cells ◽  
Phenotypic Switching ◽  
Morphological Response ◽  
Content Type ◽  
Cell Death Pathway

ABSTRACT Mating in hemiascomycete yeasts involves the secretion of pheromones that induce sexual differentiation in cells of the opposite mating type. Studies in Saccharomyces cerevisiae have revealed that a subpopulation of cells experiences cell death during exposure to pheromone. In this work, we tested whether the phenomenon of pheromone-induced death (PID) also occurs in the opportunistic pathogen Candida albicans. Mating in C. albicans is uniquely regulated by white-opaque phenotypic switching; both cell types respond to pheromone, but only opaque cells undergo the morphological transition and cell conjugation. We show that approximately 20% of opaque cells, but not white cells, of laboratory strain SC5314 experience pheromone-induced death. Furthermore, analysis of mutant strains revealed that PID was significantly reduced in strains lacking Fig1 or Fus1 transmembrane proteins that are induced during the mating process and, we now show, are necessary for efficient mating in C. albicans. The level of PID was also Ca2+ dependent, as chelation of Ca2+ ions increased cell death to almost 50% of the population. However, in contrast to S. cerevisiae PID, pheromone-induced killing of C. albicans cells was largely independent of signaling via the Ca2+-dependent protein phosphatase calcineurin, even when combined with the loss of Cmk1 and Cmk2 proteins. Finally, we demonstrate that levels of PID vary widely between clinical isolates of C. albicans, with some strains experiencing close to 70% cell death. We discuss these findings in light of the role of prodeath and prosurvival pathways operating in yeast cells undergoing the morphological response to pheromone.

scholarly journals Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl.

1995 ◽  
Vol 182 (5) ◽  
pp. 1545-1556 ◽  
Author(s):  
S J Martin ◽  
C P Reutelingsperger ◽  
A J McGahon ◽  
J A Rader ◽  
R C van Schie ◽  
...  
Keyword(s):  
Cell Death ◽  
Plasma Membrane ◽  
Programmed Cell Death ◽  
General Feature ◽  
Annexin V ◽  
Cell Types ◽  
Morphological Features ◽  
Critical Event ◽  
Specific Probe ◽  
Macromolecular Synthesis

A critical event during programmed cell death (PCD) appears to be the acquisition of plasma membrane (PM) changes that allows phagocytes to recognize and engulf these cells before they rupture. The majority of PCD seen in higher organisms exhibits strikingly similar morphological features, and this form of PCD has been termed apoptosis. The nature of the PM changes that occur on apoptotic cells remains poorly defined. In this study, we have used a phosphatidylserine (PS)-binding protein (annexin V) as a specific probe to detect redistribution of this phospholipid, which is normally confined to the inner PM leaflet, during apoptosis. Here we show that PS externalization is an early and widespread event during apoptosis of a variety of murine and human cell types, regardless of the initiating stimulus, and precedes several other events normally associated with this mode of cell death. We also report that, under conditions in which the morphological features of apoptosis were prevented (macromolecular synthesis inhibition, overexpression of Bcl-2 or Abl), the appearance of PS on the external leaflet of the PM was similarly prevented. These data are compatible with the notion that activation of an inside-outside PS translocase is an early and widespread event during apoptosis.

scholarly journals Antifungal Properties of Cationic Phenylene Ethynylenes and Their Impact on β-Glucan Exposure

2016 ◽  
Vol 60 (8) ◽  
pp. 4519-4529 ◽  
Author(s):  
Harry C. Pappas ◽  
Rina Sylejmani ◽  
Matthew S. Graus ◽  
Patrick L. Donabedian ◽  
David G. Whitten ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Membrane Integrity ◽  
Significant Risk ◽  
Yeast Cells ◽  
Size Difference ◽  
Phenylene Ethynylene ◽  
Biocidal Activity ◽  
Content Type ◽  
Antifungal Properties ◽  
Phenylene Ethynylenes

ABSTRACTCandidaspecies are the cause of many bloodstream infections through contamination of indwelling medical devices. These infections account for a 40% mortality rate, posing a significant risk to immunocompromised patients. Traditional treatments againstCandidainfections include amphotericin B and various azole treatments. Unfortunately, these treatments are associated with high toxicity, and resistant strains have become more prevalent. As a new frontier, light-activated phenylene ethynylenes have shown promising biocidal activity against Gram-positive and -negative bacterial pathogens, as well as the environmental yeastSaccharomyces cerevisiae. In this study, we monitored the viability ofCandidaspecies after treatment with a cationic conjugated polymer [poly(p-phenylene ethynylene); PPE] or oligomer [“end-only” oligo(p-phenylene ethynylene); EO-OPE] by flow cytometry in order to explore the antifungal properties of these compounds. The oligomer was found to disruptCandida albicansyeast membrane integrity independent of light activation, while PPE is able to do so only in the presence of light, allowing for some control as to the manner in which cytotoxic effects are induced. The contrast in killing efficacy between the two compounds is likely related to their size difference and their intrinsic abilities to penetrate the fungal cell wall. Unlike EO-OPE-DABCO (where DABCO is quaternized diazabicyclo[2,2,2]octane), PPE-DABCO displayed a strong propensity to associate with soluble β-glucan, which is expected to inhibit its ability to access and perturb the inner cell membrane ofCandidayeast. Furthermore, treatment with PPE-DABCO unmaskedCandida albicansβ-glucan and increased phagocytosis by Dectin-1-expressing HEK-293 cells. In summary, cationic phenylene ethynylenes show promising biocidal activity against pathogenicCandidayeast cells while also exhibiting immunostimulatory effects.

Eosinophil Cell Death Determination: Role for ERK in the Mechanism Regulating Decision Between Eosinophil Activation, Apoptosis or Regulated Necrosis.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2134-2134
Author(s):  
Gen Kano ◽  
Maha Almanan ◽  
Bruce Bochner ◽  
Nives Zimmermann
Keyword(s):  
Cell Death ◽  
Conflicts Of Interest ◽  
Membrane Integrity ◽  
Annexin V ◽  
Ros Production ◽  
Survival Factor ◽  
Cell Death Pathway ◽  
Eosinophil Cell ◽  
Regulated Necrosis ◽  
Eosinophil Activation

Abstract Abstract 2134 Siglec-8 is a membrane protein predominantly expressed on eosinophils, where its ligation induces cell death. Paradoxically, Siglec-8-induced cell death is markedly enhanced by the eosinophil activation and survival factor IL-5. Thus, Siglec-8 induces cell death preferentially in activated eosinophils, making it an attractive therapeutic target for eosinophil-mediated diseases. However, the mechanism of this survival factor-enhanced cell death is not known. While Siglec-8 ligation (by anti-Siglec-8 antibody) induces caspase-dependent apoptosis in resting eosinophils, it induces caspase-independent cell death in activated eosinophils. We hypothesize that co-stimulating the Siglec-8 and IL-5 pathways induces a necrotic cell death pathway. By morphologically characterizing human peripheral blood eosinophils as “apoptotic” (i.e., shrunk cells with condensed chromatin) or “necrotic” (i.e., swollen cells, disrupted membrane integrity), we found that anti-Siglec-8 + IL-5 co-stimulation yielded more necrotic eosinophils (P = 0.055, 6 donors) than stimulation with anti-Siglec-8 alone. Additionally, we stained with Annexin V and 7AAD and assessed the percent of Annexin V+ cells that are 7AAD+ as an indicator of increased transition of apoptotic cells to secondary necrosis and/or cells dying primarily by necrosis. We found that anti-Siglec-8 + IL-5 co-stimulated cells had a higher ratio of 7AAD+ cells compared with cells treated with anti-Siglec-8 alone (P < 0.001, 25 experiments with 11 independent donors). This higher 7AAD+ ratio, the morphological characteristics and the caspase-independent cell death of co-stimulated cells suggest that Siglec-8 ligation induces a necrotic form of cell death in IL-5-stimulated eosinophils by activating a specific and distinct biochemical pathway. Our previous studies have shown that reactive oxygen species (ROS) production is involved in Siglec-8-induced cell death in both resting and activated eosinophils. However, we have observed that phosphorylation of ERK1/2 and MEK1 was significantly increased in cells co-stimulated with anti-Siglec-8 + IL-5 compared to cells stimulated with IL-5 alone; anti-Siglec-8 alone did not cause ERK1/2 phosphorylation. MEK1 inhibitors U0126 and PD184352 completely blocked anti-Siglec-8 + IL-5-induced cell death; however, intracellular ROS production induced by Siglec-8 ligation was MEK1-independent. In contrast, the ROS inhibitor DPI prevented the anti-Siglec-8 + IL-5-induced enhancement of ERK1/2 phosphorylation and subsequent cell death. Enhanced ROS accumulation in IL-5 treated cells was sufficient to induce enhanced cell death, similar to anti-Siglec-8 treatment. These findings suggest that Siglec-8 ligation leads to ROS-dependent enhancement of IL-5-induced ERK1/2 phosphorylation, which results in enhanced Siglec-8-induced eosinophil cell death. How ERK phosphorylation induces cell death in co-stimulated eosinophils is not known, and ERK's involvement is surprising considering its role in activation of IL-5-stimulated eosinophils. However, recent studies have shown that ERK can be involved in specific types of cell death, namely necroptosis or autophagy, and that spatiotemporal parameters determine whether ERK will induce cell death or activation. Thus, we hypothesized that ERK localization will be altered in eosinophils co-stimulated with anti-Siglec-8 + IL-5 compared with cells treated with IL-5 alone. Western blotting of nuclear and cytoplasmic fractions and immunofluorescence suggest that enhanced ERK1/2 localization and phosphorylation are sustained for at least 2 hours in the nucleus of anti-Siglec-8 + IL-5 co-stimulated cells; cells treated with IL-5 alone have only brief ERK1/2 nuclear localization. The sustained nuclear activation of ERK may explain the change in IL-5 function from eosinophil activation/survival to necrotic death upon Siglec-8 ligation. In summary, ERK is involved in regulating the decision point for eosinophil activation, apoptosis or regulated necrosis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Critical Evaluation of Techniques to Detect and Measure Cell Death – Study in a Model of UV Radiation of the Leukaemic Cell Line HL60

1999 ◽  
Vol 19 (3-4) ◽  
pp. 139-151 ◽  
Author(s):  
Marina Leite ◽  
Margarida Quinta‐Costa ◽  
Pedro Simas Leite ◽  
José Eduardo Guimarães
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Fluorescence Microscopy ◽  
Trypan Blue ◽  
Critical Evaluation ◽  
Annexin V ◽  
Tunel Assay ◽  
Apoptotic Cells ◽  
Dna Ladder ◽  
Apoptosis And Necrosis

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.

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