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In the current investigation, watermelon rinds (WMR) have been utilized as an eco-friendly and cost-efficient adsorbent for acridine orange (AO) from contaminated water samples. Adsorption of AO onto raw (RWM) and thermally treated rinds (TTWM250 and TTWM500) has been studied. The adsorption efficiency of the three adsorbents was evaluated by measuring the % removal (%R) of AO and the adsorption capacity (q e , mg/g). Dependent variables (%R and q e ) were optimized as a function of four factors: pH, sorbent dosage (AD), the concentration of AO (DC), and contact time (ST). Box-Behnken (BB) design has been utilized to obtain the optimum adsorption conditions. Prepared adsorbents have been characterized using scanning electron microscopy (SEM), Fourier-transform infrared (FT-IR), and Raman spectroscopies. The surface area of RWM, TTWM250, and TTWM500, as per the Brunauer-Emmett-Teller (BET) analysis, was 2.66, 2.93, and 5.03 m 2 /g, respectively. Equilibrium investigations suggest that Freundlich model was perfectly fit for adsorption of AO onto TTWM500. Maximum adsorption capacity (q max ) of 69.44 mg/g was obtained using the Langmuir equation. Adsorption kinetics could be best described by the pseudo-second-order (PSO) model. The multi-cycle sorption-desorption study showed that TTWM500 could be regenerated with the adsorption efficiency being preserved up to 87% after six cycles., (© 2021. The Author(s).)

The perfluorooctane sulfonate alternative, F-53B, induces multiple physiological defects but whether it can disrupt eye development is unknown. We exposed zebrafish to F-53B at four different concentrations (0, 0.15, 1.5, and 15 μg/L) for 120 h post-fertilization (hpf). Locomotor behavior, neurotransmitters content, histopathological alterations, morphological changes, cell apoptosis, and retinoic acid signaling were studied. Histology and morphological analyses showed that F-53B induced pathological changes in lens and retina of larvae and eye size were significantly reduced as compared to control. Acridine orange (AO) staining revealed a dose-dependent increase in early apoptosis, accompanied by upregulation of p53, casp-9 and casp-3 genes. Genes related to retinoic acid signaling (aldh1a2), lens developmental (cryaa, crybb, crygn, and mipa) and retinal development (pax6, rx1, gant1, rho, opn1sw and opn1lw) were significantly downregulated. In addition, behavioral responses (swimming speed) were significantly increased, while no significant changes in the neurotransmitters (dopamine and acetylcholine) level were observed. Therefore, in this study we observed that exposure to F-53B inflicted histological and morphological changes in zebrafish larvae eye, induced visual motor dysfunctions, perturbed retinoid signaling and retinal development and ultimately triggering apoptosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The adsorption of acridine orange and Cr 6+ ion onto plaster of paris reinforced glutamic acid-grafted-polyacrylamide hydrogel nanocomposite modified with riboflavin, Glu-g-PAM/POP/Rb HNC was studied. The Glu-g-PAM/POP/Rb HNC was physico-chemically characterized by Fourier transform infrared spectroscopy, X-ray diffraction analysis, scanning electron microscopy coupled with energy dispersive X-ray spectroscopy, transmission electron microscopy and Brunauer-Emmett-Teller analysis. The specific surface area, pore volume and pore diameter were 15.48 m 2 /g, 0.015 cm 3 /g and 4.23 nm, respectively. Adsorption process was strategized by response surface methodology (RSM) based on a 3-level 5-factor (initial solution pH, contact time, adsorbent dose, initial adsorbate concentration and temperature) central composite design (CCD), and validity of the estimated parameters was statistically evaluated using analysis of variance (ANOVA). The optimized operating variables were: pH (AO = 10; Cr 6+  = 4.15), contact time (AO = 60 min; Cr 6+  = 59 min), adsorbent dose (0.8 g/L), initial adsorbate concentration (60 mg/L) and temperature (298 K). Isotherm results were coincident with Langmuir isotherm model. The experimental kinetic adsorption data was congruous with pseudo-second order model, with the uptake rate controlled by both intraparticle and liquid film diffusions. The relatively high Langmuir saturation capacity of 202.63 mg AO/g and 143.68 mg Cr 6+ /g, supported by the decent recyclability up to four times affirmed the promising performance of the adsorbent. The efficacy of the adsorbent for simultaneous removal of AO and Cr 6+ from bi-component system was assessed. The possible adsorption mechanism mainly involved hydrogen bonding, van der Waals forces, electrostatic and π-π interactions. Adsorption of AO and Cr 6+ onto Glu-g-PAM/POP/Rb HNC was feasible and exothermic as revealed by the thermodynamic parameters. The findings demonstrated superior adsorbent efficacy for the seizure of pollutants, particularly AO and Cr 6+ from aqueous solution., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Objectives: Although halophilic archaea are rich in natural environments, their biotechnological applications are not as prevalent as those of other extremophiles, such as thermophiles and alkaliphiles. This study presents an simple method to prepare a hydrogel composite using crude cell lysate of a halophilic archaea, Halorubrum ejinoor sp. (H.e.) which was isolated from a saline lake in Inner Mongolia, China. Furthermore, formation mechanism and potential applications of the hydrogel as an adsorbing material are discussed., Results: Halorubrum ejinoor sp. (H.e.) cell lysate was firstly prepared by adding pure water onto the H.e. cell pellet, followed by a short incubation at 60 °C. The cell lysate was injected into different metal ion (or H + ) solutions to obtain the hydrogel composite. It was observed that H + , Fe 3+ , La 3+ , Cu 2+ , and Ca 2+ induced gelation of the cell lysate, while Fe 2+ , Co 2+ , Ni 2+ , Mg 2+ , Na + , and K + did not. DNA and extracellular polysaccharides (EPS) in the H.e. cell lysate were found to be responsible for the gelation reaction. These results suggest that DNA and EPS should be crosslinked by metal ions (or H + ) and form a networked structure in which the metal ion (or H + ) serves as an anchor point. Potential application of the hydrogel as an adsorbing material was explored using La 3+ -induced H.e. hydrogel composite. The hydrogel composite can adsorb the fluoride, phosphate and DNA-binding carcinogenic agents, such as acridine orange., Conclusions: The simplicity and cost effectiveness of the preparation method might make H.e. hydrogel a promising adsorbing material. This work is expected to expand the technical applications of haloarchaea.

Programmed cell death (PCD) processes are essential in the plant embryogenesis. To understand how PCD operates in a developing seed, the dying cells need to be identified in relation to their surviving neighbors. This can be accomplished by the means of in situ visualization of fragmented DNA-a well-known hallmark of PCD. In the developing Scots pine (Pinus sylvestris L.) seed, several tissues die via morphologically different PCD processes during the embryogenesis. Here, we describe the protocols for the characterization of Scots pine seeds at the early and late developmental stages and, further, the localization of nucleic acids and DNA fragmentation by the acridine orange staining and TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labeling) assay in the dying seed tissues.

Quantitative assessment of changes in macro-autophagy is often performed through manual quantification of the number of LC3B foci in immunofluorescence microscopy images. This method is highly laborious, subject to image-field selection and foci-counting bias, and is not sensitive for analyzing changes in basal autophagy. Alternative methods such as flow cytometry and transmission electron microscopy require highly specialized, expensive instruments and time-consuming sample preparation. Immunoblots are prone to exposure-related variations and noise that prevents accurate quantification. We report a high-throughput, inexpensive, reliable and objective method for studying basal level and flux changes in late-stage autophagy using image cytometry and acridine orange staining.

Based on the fluorescent reversible regulation, a novel sensor platform was designed for the detection of DNA intercalators utilizing the intercalation binding of DNA intercalators to DNA as an inherent exhibition and the fluorescence change of quantum dots (QDs) as an external manifestation. To prove its feasibility, acridine orange (AO) was chosen as an example of DNA intercalator. When different concentrations of herring sperm DNA (hsDNA) were added to cysteamine (CA)-capped ZnSe QDs solution, the hsDNA bound with the QDs through electrostatic interaction due to the photoinduced electron transfer from hsDNA to QDs and formed QDs-hsDNA complexes with 1:1 ratio, leading to the fluorescence quenching of the QDs; and upon addition of different concentrations of AO to the QDs-hsDNA complex system, the AO first caused the release of the hsDNA from the complexes and concomitantly bound with them through intercalation binding and formed AO-hsDNA complexes with 1:3 ratio on account of the fact that the intercalation binding constant between AO and hsDNA (1.932 × 10 5 L/mol) was greater than the electrostatic interaction constant between QDs and hsDNA (7.874 × 10 4 L/mol), resulting in the fluorescence recovery of the QDs. Therefore, the detection of AO could be achieved through the relationship between the fluorescence recovery yield of the QDs and the concentration of AO added. The results illustrated that the fluorescence recovery yield of the QDs-hsDNA system was linearly dependent to the concentration of AO in the range of 5.0-75.0 × 10 -5 mol/L with a detection limit (3σ/K) of 1.5 × 10 -5 mol/L. This dual-directional fluorescent regulation provided a novel method for the detection of DNA intercalators such as polycyclic aromatic hydrocarbons and drugs interfering with DNA-synthesis and possessed some potential applications in the investigation of the interactions between DNA intercalators and DNA., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Giant plasma membrane vesicles (GPMVs) are large extracellular vesicles produced by the exposure of cells to paraformaldehyde or other stresses, providing an experimental system to elucidate cell surface dynamics. Here we show that addition of the membrane permeable fluorescent RNA-indicators, acridine orange and thioflavin T, to GPMV-containing solutions prepared from cultured HeLa cells was sufficient for the fluorescent visualization of seemingly all GPMVs. Our findings provide a wash-free instant method using non-lipid-type fluorescent dyes for GPMV detection, which should be useful for researchers interested in studying cell membrane dynamics and biochemistry using GPMVs., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A series of optimized protocols to isolate vacuoles from both yeast and plant cells, and to characterize the purified organelles at a functional and structural level, are described. For this purpose, we took advantage of the combined use of cell fractionation techniques with different fluorescence-based approaches namely flow cytometry, fluorescence microscopy and spectrofluorimetry. These protocols altogether constitute valuable tools for the study of vacuole structure and function, as well as for the high-throughput screening of drug libraries to identify new molecules that target the vacuole.

The fluorescence spectrum of bacterially bound acridine orange (AO) was investigated to evaluate its use for the rapid enumeration of bacteria. Escherichia coli ATCC 25922 samples were stained with 2 × 10 -2 , 2 × 10 -3 or 2 × 10 -4 % w/v AO, followed by 3, 2 or 0 washing cycles, respectively, and fluorescence spectra were recorded using a fibre-based spectroscopic system. Independent component analysis was used to analyse the spectral datasets for each staining method. Bacterial concentration order of magnitude classification models were calculated using independent component weights. The relationship between fluorescence intensity of bound AO and bacterial concentration was not linear. However, the spectral signals collected for AO stain concentration-bacterial concentration pairs were reproducible and unique enough to enable classification of samples. When above 10 5  CFU ml -1 , it was possible to rapidly determine what the order of magnitude of bacterial concentration of a sample was using a combination of two of the sample preparation methods. A relatively inexpensive (around US$10 per test) rapid method (within 25 min of sampling) for enumeration of bacteria by order of magnitude will reduce the time and cost of microbiological tests requiring gross concentration information. Graphical Abstract Fluorescence spectra of bacterially bound acridine orange (AO) were used for the rapid enumeration of bacteria. Order of magnitude bacterial concentration classification models were calculated using independent components analysis of these fluorescence spectra. When above 10 5 CFU ml -1 , it was possible to rapidly determine the order of magnitude of bacterial concentration of a sample using a combination of two sample preparation methods.

RNA interference (RNAi) is an effective tool to study gene function. For in vitro studies of RNAi in insects, microinjection of double-stranded (ds)RNA may cause stress. Non-persuasive oral delivery of dsRNA to trigger RNAi is a better mode of delivery for delicate insects such as aphids because it mimics natural feeding. However, when insects feed ad libitum, some individuals may not feed. For accurate measurement of gene knockdown, analysis should only include insects that have ingested dsRNA. The suitability of eleven dyes was assessed to trace ingestion of dsRNA in an artificial feeding system for green peach aphids (GPA, Myzus persicae ). Non-toxic levels of neutral red and acridine orange were suitable tracers: they were visible in the stylet and gut after feeding for 24 h, and may also attract aphids to feed. Nymphs stained with neutral red (0.02%) were analysed for target gene expression after feeding on sucrose with dsRNA (V-ATPase, vha-8 ). There was a greater reduction in vha-8 expression and reproduction compared to nymphs fed the diet without dye. The results confirm the importance of identifying aphids that have ingested dsRNA, and also provide evidence that the vha-8 gene is a potential target for control of GPAs., Competing Interests: The authors declare no conflict of interest.

Heavy metals such as mercury (Hg) pose a significant health hazard through bioaccumulation and biomagnification. By penetrating cell membranes, heavy metal ions may lead to pathological conditions. Here we examined the responses of Ammonia parkinsoniana, a benthic foraminiferan, to different concentrations of Hg in the artificial sea water. Confocal images of untreated and treated specimens using fluorescent probes (Nile Red and Acridine Orange) provided an opportunity for visualizing the intracellular lipid accumulation and acidic compartment regulation. With increased Hg over time, we observed an increased number of lipid droplets, which may have acted as a detoxifying organelle where Hg is sequestered and biologically inactivated. Further, Hg seems to promote the proliferation of lysosomes both in terms of number and dimension that, at the highest level of Hg, resulted in cell death. We report, for the first time, the presence of Hg within the foraminiferal cell: at the basal part of pores, in the organic linings of the foramen/septa, and as cytoplasmic accumulations., Competing Interests: The authors have declared that no competing interests exist.

Fe3O4 nanoparticles were grown in situ on carbon nanotubes (CNTs) by a solvothermal method. The Fe3O4/CNTs composites were characterised by the Brunauer-Emmett-Teller method and transmission electron microscopy. The results indicated that the Fe3O4 nanoparticles were uniformly deposited on CNTs, and the average diameter was approximately 7.0 nm. The Fe3O4/CNTs were applied as an enzyme mimetic to decompose Orange II, and the decomposing conditions were optimised. At 500 mg L(-1) of Fe3O4/CNTs in the presence of 15.0 mmol L(-1) of H2O2, at 30°C, it degraded 94.0% of Orange II (0.25 mmol L(-1), pH = 3.5), showing higher catalytic activity than pure Fe3O4 nanoparticles. The high activity was attributed to the uniform Fe3O4 nanoparticles growing on the side walls of the CNTs and the synergetic effect between Fe3O4 and CNTs. The Fe3O4/CNTs maintained their activity at temperatures as high as 65°C. The Fe3O4/CNTs presented high reusability and stability even after eight uses. These data proved that the Fe3O4/CNTs-catalysed degradation is a promising technique for wastewater treatment. Fe3O4 nanoparticles were grown in situ on carbon nanotubes (CNTs) by a solvothermal method. The Fe3O4/CNTs was applied as a mimetic enzyme to decompose Orange II. The Fe3O4/CNTs were collected after the reaction by applying an external magnetic field and can use repeatedly.

Background: Dilution bias is a major cause of immunoassay variability due to the lack of an internal standard to determine the true versus the expected dilution value., Methodology: We used an internal control to measure dilution bias in an ELISA. Acridine-orange was added at the first dilution step and monitored throughout dilutions. Assay results were corrected using the fluorescent signal ratio between samples and reference. Acridine dilution correlated with analyte-specific assay measurements (R2 = 0.987). Correction of assay results with the measured dilution factor improved both accuracy and precision resulting in a reduction of >50% %CV reduction., Conclusion: Dilution correction can significantly improve accuracy and precision of immunoassays. Additional control strategies may further mitigate other sources of variability.

A novel dye-based method for measuring cell death in image-based screens is presented. Unlike conventional high- and medium-throughput cell death assays that measure only one form of cell death accurately, using multivariate analysis of micrographs of cells stained with the inexpensive mix, red dye nonyl acridine orange, and a nuclear stain, it was possible to quantify cell death induced by a variety of different agonists even without a positive control. Surprisingly, using a single known cytotoxic agent as a positive control for training a multivariate classifier allowed accurate quantification of cytotoxicity for mechanistically unrelated compounds enabling generation of dose-response curves. Comparison with low throughput biochemical methods suggested that cell death was accurately distinguished from cell stress induced by low concentrations of the bioactive compounds Tunicamycin and Brefeldin A. High-throughput image-based format analyses of more than 300 kinase inhibitors correctly identified 11 as cytotoxic with only 1 false positive. The simplicity and robustness of this dye-based assay makes it particularly suited to live cell screening for toxic compounds.

The morphological evolutions of orthorhombic molybdenum oxide nanostructures with high crystalline nature have been successfully synthesized by combining low-temperature sol-gel and annealing processes. Strong influence of gelation temperature is a factor facilitated to control the material morphology. Morphological transformations like nanospheres, nanoplatelets, mixtures of hexagonal platelets, and one-dimensional nanobars were obtained. The possible morphological formation mechanism has been proposed as a self-assemble process of nucleation and a mechanism for particle growth by Ostwald ripening. The as-prepared nanostructures were recognized as photocatalysts for the degradation of Acridine Orange under Ultra Violet light. The obtained mixed morphology (hexagonal nanoplatelets and nanobars) showed a high photocatalytic property to degrade mutagenic Acridine Orange dye. Moreover, they could be easily recycled without changing the photocatalytic activity due to their 1-Dimensional and 2-Dimensional nanostructure property.

Documentation of Helicobacter pylori infection and eradication is important, prompting some clinicians and pathologists to request ancillary stains on all gastric samples that do not demonstrate H. pylori on initial histologic review. Studies evaluating the utility of ancillary stains in patients with minimal inflammation are lacking. We used Giemsa, Warthin-Starry, acridine orange, and immunohistochemical stains to search for organisms in 56 patients with biochemical evidence of H. pylori infection (positive Campylobacter-like organism test) and gastric mucosal samples interpreted to be H pylori negative by hematoxylin and eosin (H&E). We correlated the findings with severity of inflammation and patients' histories of medication use. Nineteen (34%) patients had histologically normal mucosae, 22 (39%) had chronic inflammation with or without focal activity, and 15 (27%) had chemical gastropathy. Fifty (89%) cases were negative for H. pylori with additional stains, and 6 contained bacteria that were detected with all 4 ancillary stains and on retrospective review of H&E-stained sections that also showed chronic inflammation. Eleven (20%) patients were taking proton pump inhibitors, and 4 (7%) had previously received H. pylori eradication therapy. We conclude that H&E stains demonstrate H. pylori in most infected patients, so preemptive stain requests are largely unnecessary. Failure to identify bacteria by H&E evaluation generally reflects their absence in biopsy material, even among Campylobacter-like organism test--positive patients. However, organisms may be overlooked in patients with mild inflammation and in those receiving proton pump inhibitor or antibiotic therapy, so one should consider ordering ancillary stains to enhance detection of bacteria in these settings., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: We have previously developed an affordable flow cytometric method for absolute cell count using glutaraldehyde-fixed chicken red blood cells. However, its use is limited to CD4+ T cells. In the current investigation, we studied the potential use of glutaraldehyde-fixed chicken RBCs to determine the number of residual white blood cells (rWBCs) in WBC-reduced blood component., Methods: Acridine orange (AO) was used to identify leucocytes in serial diluted blood samples ranging from 0.65 to 1,000 cells/microL. The absolute number of AO stained leucocytes were determined by using a known number of glutaraldehyde-fixed chicken RBCs on flow cytometer. The results were compared with the expected value and the absolute count determined by BD Leucocount (Becton Dickinson Bioscience). In addition, the stability of AO stained leucocytes and sample stability at various time points were measured. Reproducibility of the assay method was also addressed., Results: There was a good correlation in the number of leucocytes between our new method and the expected numbers from serially diluted blood samples (r2 = 0.99, y = 1.04x + 0.50, p < 0.001). Furthermore, absolute leucocyte counts determined by the new method correlated well with those obtained from BD Leucocount (r2 = 0.99, y = 1.31 x - 6.37, p < 0.001). FL-1 intensity and the absolute number of AO stained leucocytes were stable for at least 24 hours after staining. Samples stored at 4 degrees C were stable for 48 hours and CV of the assay was at an acceptable level., Conclusion: This flow cytometric method for absolute leucocyte counts using AO and glutaraldehyde-fixed chicken RBCs is a simple, rapid, reliable and inexpensive method for routine monitoring of low levels of leucocytes in blood products.

Synthesis, molecular structure and cytotoxic activity of a series of 3-C, N, S, Se substituted benzo[b]selenophene derivatives on human fibrosarcoma HT-1080, mouse hepatoma MG-22A, and mouse fibroblasts 3T3 cell lines are described. The correlation between compound LD(50) 3T3 fibroblast cell line and HT-1080 morphology was shown., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

The antitumor effects and molecular mechanism of NPC-16, a novel naphthalimide-polyamine conjugate, were evaluated in HepG2 cells and Bel-7402 cells. Apoptosis and necrosis were evaluated by Annexin V-FITC detection kit, and autophagy by acridine orange and Lyso-Tracker Red staining. The change of mitochondrial transmembrane potential was measured using rhodamine 123 staining. The protein expression of Beclin 1, LC3 II and mTOR, p70S6 K, 14-3-3, caspase, and Bcl-2 family members was detected by immunofluorescence assays and Western Blot. Here, we elucidated the nature of cellular response of HepG2 cells and Bel-7402 cells to NPC-16 at IC(50). NPC-16 induced caspase-dependent apoptosis via the mitochondrial pathway and death receptor pathway in Bel-7402 cells. Differently, NPC-16 triggered HepG2 cells both apoptosis and autophagy, further autophagy facilitated cellular apoptosis. Furthermore, mTOR signal pathway was involved in NPC-16-mediated autophagy in HepG2 cells. Thus, NPC-16 may be useful as a potential template for investigation the molecular mechanism of naphthalimide-polyamine conjugate against hepatocellular carcinoma.

Time course of changes in intracellular water, K+ and Na+ of U937 cells incubated in hyperosmolar medium with addition of 200 mM sucrose was studied. Ouabain-sensitive and ouabain-resistant Rb+ (K+) influxes were measured during regulatory cell volume increase (RVI) and apoptotic volume decrease (AVD). Microscopy of cells stained by Acrydine orange, Ethydium bromide, APOPercenrage Dye and polycaspase marker FLICA was performed. We found that initial osmotic cell shrinkage induced both RVI and AVD responses. RVI dominated at the early stage whereas AVD prevailed at the later stage. In view of the data obtained in U937 cells the current opinion that RVI "dysfunction" is a prerequisite for apoptosis and AVD (Subramanyam et al., 2010) should be revised. U937 cells are capable to trigger of apoptosis and AVD in spite of the unimpaired RVI response. It is concluded that AVD plays a significant role in preventing osmotic lysis of apoptotic cells rather than in the initiation of apoptosis.

The interaction of acridine orange with blood albumins and tissue cells from different organs of white mouse has been studied by the spectral luminescence method. It was shown that acridine orange, by penetrating cells or orangelles, is able to intercalate between base pairs in the DNA molecule. It was found that the application of acridine orange as a fluorescent probe can influence the metabolic activity of organs.

The present study investigates the potential use of haemocyte lysosomal fragility as a reliable indicator of toxic injury in snails subjected to herbicide atrazine. Two cytochemical assays were used. The first one was based on microscopic observations of neutral red dye, and the second one on a fluorescence spectroscopy approach using acridine orange, which had the advantage of being more reliable in assessing minor cell damage. Two sets of experiments were carried out in controlled laboratory conditions (temperature, photoperiod) and in outdoor microcosms, which produce more realistic ecological conditions. An unequivocal relationship was shown, linking atrazine exposure to Lymnaea stagnalis haemocytes lysosomal permeability. High temperatures also had a negative effect on the lysosomal membrane. In the microcosms, the response was affected by seasonal factors, and led to an integrated cell response. It was concluded that lysosomal sensitivity reflects environmental changes. Moreover, the technique using acridine orange was shown to be sufficiently sensitive and reliable to serve as a lysosomal marker in a controlled environment.

Aim: To develop express method for detection of neutrophil extracellular traps represented by extracellular strands of neutrophils' DNA., Materials and Methods: Acridine orange staining was used for visualization of neutrophil exracellular traps, which allows to quickly reveal the forming structures and to quantify this phenomenon., Results: Performed studies showed that after 30-min of in vitro interaction of Escherichia coli, Staphylococcus aureus, lactobacteria, bifidobacteria and latex particles with neutrophils, the latter produces extracellular net-like structures, which were well visualized after staining of fixed preparations with acridine orange. Such structures are able to trap latex particles and bacteria more efficiently than live cell. Additionally, representatives of normal flora (bifidobacteria) actively stimulate production of traps., Conclusion: Production of neutrophil extracellular traps could be one of the main and effective mechanisms in antimicrobial defense of mucosal surfaces.

Using spectral scanning regime of Leica TCS SL confocal microscope, acridine orange (AO) fluorescence spectra in nuclei and cytoplasms of living myoblasts L6J1 and frog single muscle fibres have been studied. AO fluorescence spectra in salt solutions dependent on free AO concentrations and in AO complexes with DNA have also been obtained for comparison. Myoblasts nuclei fluoresced in green spectral region with maximum at approximately 530 nm (corresponding AO monomers fluorescence), nucleoli fluoresced most brightly. Nuclear chromatin fluoresced not uniformly in these cells. We saw similar to myoblasts AO emission in nucleoli and nuclei of frog single muscle fibres. The uniformed weak green fluorescence was observed for myoblast cytoplasm. As to the muscle fibres sarcoplasm, we saw also AO green fluorescence in A-discs. In myoblasts and muscle fibre cytoplasm we saw the fluorescent red, yellow and green granules which were acidic organelles. The comparison of AO fluorescence spectra in living cells with fluorescence spectra of different AO concentrations and complexes of AO with DNA in buffer solutions allows estimation of AO concentration in acidic granules which is of interest in the investigation of cellular organelles functions in the processes of intracellular transport, adaptation, apoptosis and a number of pathological conditions.

Based on the reaction of acridine orange with deoxyribonucleic acid (DNA), and under the optimized condition, a novel method to determine the DNA was developed with the spectrophotometry. The absorbance of acridine orange at the maximum absorption wavelength of 444 nm responded to the concentration of DNA negatively with excellent linearity. It has an upper linear limit concentration of 8.0 microg x mL(-1) and a detection limit of 0.12 microg x mL(-1) with the correlation coefficient of 0.999 8. The method is simple, rapid and sensitive, and could be applied to sample assay satisfactorily. Finally, the reaction mechanism is discussed.

Previous investigations of the salinity effects on the microbial community composition have largely been limited to dynamic estuaries and coastal solar salterns. In this study, the effects of salinity and mineralogy on microbial community composition was studied by using a 900-cm sediment core collected from a stable, inland hypersaline lake, Lake Chaka, on the Tibetan Plateau, north-western China. This core, spanning a time of 17,000 years, was unique in that it possessed an entire range of salinity from freshwater clays and silty sands at the bottom to gypsum and glauberite in the middle, to halite at the top. Bacterial and archaeal communities were studied along the length of this core using an integrated approach combining mineralogy and geochemistry, molecular microbiology (16S rRNA gene analysis and quantitative polymerase chain reaction), cultivation and lipid biomarker analyses. Systematic changes in microbial community composition were correlated with the salinity gradient, but not with mineralogy. Bacterial community was dominated by the Firmicutes-related environmental sequences and known species (including sulfate-reducing bacteria) in the freshwater sediments at the bottom, but by halophilic and halotolerant Betaproteobacteria and Bacteroidetes in the hypersaline sediments at the top. Succession of proteobacterial groups along the salinity gradient, typically observed in free-living bacterial communities, was not observed in the sediment-associated community. Among Archaea, the Crenarchaeota were predominant in the bottom freshwater sediments, but the halophilic Halobacteriales of the Euryarchaeota was the most important group in the hypersaline sediments. Multiple isolates were obtained along the whole length of the core, and their salinity tolerance was consistent with the geochemical conditions. Iron-reducing bacteria were isolated in the freshwater sediments, which were capable of reducing structural Fe(III) in the Fe(III)-rich clay minerals predominant in the source sediment. These data have important implications for understanding how microorganisms respond to increased salinity in stable, inland water bodies.

The aim of this study was to investigate the viable but nonculturable (VBNC) state of the bacterium. Vibrio alginolyticus VIB283 was cultured in sterilized seawater microcosm at 4 degrees C. Culturability of the cells in the microcosm was monitored by spread plate count (PC) on 2216E agar, PCs declined to undetectable levels (<0.1 CFU/ml) within 90 days. Total cell counts remained constant throughout the period as determined by acridine orange direct count (AODC). The direct viable counts, on the other hand, declined from 10(10) to 10(9) CFU/ml active cells and remained fairly constant at this level by direct viable count (DVC), which indicated that a large population of cells entered into the VBNC state. The VBNC cells could be resuscitated by temperature upshift with and without the presence of nutrition. The resuscitated time were 16 h and 8 days respectively. The resuscitation was not achieved in chick embryos. The morphology of the VBNC, normal and resuscitated cells was studied with scanning electron microscope and flow cytometry. The cells changed from rod or arc to coccoid and decreased in size when entered into the VBNC state. The resuscitated and the normal cells had almost no morphological differences.

Dual-color imaging of acridine orange (AO) and EGFP fused to a vesicular glutamate transporter or the vesicle-associated membrane proteins 2 or 3 has been used to visualize a supposedly well-defined subpopulation of glutamatergic astrocytic secretory vesicles undergoing regulated exocytosis. However, AO metachromasy results in the concomitant emission of green and red fluorescence from AO-stained tissue. Therefore, the question arises whether AO and EGFP fluorescence can be distinguished reliably. We used evanescent-field imaging with spectral fluorescence detection as well as fluorescence lifetime imaging microscopy to demonstrate that green fluorescent AO monomers inevitably coexist with red fluorescing AO dimers, at the level of single astroglial vesicles. The green monomer emission spectrally overlaps with that of EGFP and produces a false apparent colocalization on dual-color images. On fluorophore abundance maps calculated from spectrally resolved and unmixed single-vesicle spectral image stacks, EGFP is obscured by the strong green monomer fluorescence, precluding the detection of EGFP. Hence, extreme caution is required when deriving quantitative colocalization information from images of dim fluorescing EGFP-tagged organelles colabeled with bright and broadly emitting dyes like AO. We finally introduce FM4-64/EGFP dual-color imaging as a remedy for imaging a distinct population of astroglial fusion-competent secretory vesicles.

The ability to easily analyze apoptosis is important in studies of molecular cell biology and to evaluate the relative toxicity of different treatments or environments. This is particularly the case when substances such as morpholino oligonucleotides are injected into embryos, as such treatments can cause widespread, complex patterns of apoptosis. Zebrafish embryos are well suited for cell biological and environmental toxicity analyses, but the need remains for a simple method that can analyze levels of apoptosis in a statistically significant number of embryos. Here we present a "group fluorescence" method for rapid, large-scale analysis of relative levels of apoptosis based on densitometric techniques.

Background: The aim of this study was to determine if only sperm with double stranded DNA could bind to the human zona pellucida (ZP)., Methods: Sperm samples from 124 infertile men with a range of semen abnormalities were studied. Oocytes that had failed to fertilize in IVF or ICSI were used for the sperm-ZP binding test. A group of four oocytes were incubated for 2 h with 2 x 10(6)/ml motile sperm selected by colloidal silica gradient centrifugation (CSGC). After assessing the number of sperm bound per ZP, all sperm bound to the surface of the ZP of four oocytes were dislodged and placed on a glass slide. The double (green fluorescence) or single stranded (denatured, red fluorescence) DNA of sperm in semen, motile sperm selected by CSGC and ZP-bound sperm, was assessed by acridine orange (AO) fluorescence., Results: The percentage of sperm with green fluorescence was significantly correlated with normal sperm morphology in semen and after CSGC preparation. The proportion of sperm with green fluorescence was significantly higher in motile sperm selected by CSGC than ejaculated sperm. There were very few sperm (average <8%) with red fluorescence bound to the ZP, even in men who had very high (>70%) proportions of sperm with red fluorescence in their semen., Conclusion: Sperm binding to human ZP is highly selective for double stranded DNA. Sperm with single stranded or denatured DNA bind less or do not bind at all to the ZP, probably because of defects of motility and, more especially, morphology.

The interaction of acridine orange (AO) and heparin was studied by spectrophotometry. In the pH 2. 0 Britton-Robinson (B-R) buffer solution, AO had a maximum absorption at 478 nm and 492 nm. After the addition of heparin, the absorbance value at 492 nm decreased greatly and a new absorption peak at 453 nm appeared, indicating a strong interaction taking place in the reaction solution. Under the optimal condition, the decrease in absorbance at 492 nm was linear with the concentration of heparin in the range of 3. 0-15.0 mg m L(-1). The m olar absorptivity w as calculated to be E=l. 599X 10(5) L mol(-1) x cm(-1) and the detection limit was 0. 236 mg x L(-1) (3sigma). The method was furthur applied to the detection of heparin sodium injection samples with satisfactory results. The binding number was calculated to be 1 : 3 with molar ratio method.

Bulk studies are not suitable to describe and study cell-to-cell variation, which is of high importance in biological processes such as embryogenesis, tissue differentiation, and disease. Previously, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was used to measure the properties of organelles isolated from millions of cells. As such, these bulk measurements reported average properties for the organelles of cell populations. Similar measurements for organelles released from single cells would be highly relevant to describe the subcellular variations among cells. Toward this goal, here we introduce an approach to analyze the mitochondria released from single mammalian cells. Osteosarcoma 143B cells are labeled with either the fluorescent mitochondrion-specific 10-N-nonyl acridine orange (NAO) or via expression of the fluorescent protein DsRed2. Subsequently, a single cell is introduced into the CE-LIF capillary where the organelles are released by a combined treatment of digitonin and trypsin. After this treatment, an electric field is applied and the released organelles electromigrate toward the LIF detector. From an electropherogram, the number of detected events per cell, their individual electrophoretic mobilities, and their individual fluorescence intensities are calculated. The results obtained from DsRed2 labeling, which is retained in intact mitochondria, and NAO labeling, which labels all mitochondria, are the basis for discussion of the strengths and limitations of this single-cell approach.

The data concerning the mutagenic, clastogenic and carcinogenic properties of inorganic lead compounds have been conflicting. Here, we evaluated the frequency of micronuclei in the peripheral blood of female rats treated with three different lead acetate doses. Outbred female Wistar rats were treated by gavage once per week for 10 weeks with cumulative doses of 140, 250 and 500 mg/kg body weight (body wt) of lead acetate. Mitomycin C (MMC) 2 mg/kg body wt was used as a positive control. The aim of the present study was to investigate the possible cytotoxic and genotoxic effects of lead acetate on peripheral blood reticulocytes using the micronucleus test following chronic exposure. The results show the effects of lead acetate in peripheral blood reticulocytes. These effects are both cytotoxic and genotoxic because of a decrease in the number of polychromatic erythrocytes in the peripheral blood and an increase in frequency of micronucleated reticulocytes, respectively.

Opening of individual exocytotic fusion pores in chromaffin cells was imaged electrochemically with high time resolution. Electrochemical detector arrays that consist of four platinum microelectrodes were microfabricated on a glass coverslip. Exocytosis of single vesicles containing catecholamines from a cell positioned on top of the array is detected by the individual electrodes as a time-resolved oxidation current, reflecting the time course of arrival of catecholamine molecules at the electrode surfaces. The signals exhibit low noise and reveal foot signals indicating fusion pore formation and expansion. The position of individual release events is determined from the fraction of catecholamines recorded by the individual electrodes. Simultaneous fluorescence imaging of release of acridine orange from individual vesicles confirmed the electrochemical position assignments. This electrochemical camera provides very high time resolution, spatiotemporal localization of individual fusion pore openings and quantitative data on the flux of transmitter from individual vesicles. Analysis of the amperometric currents employing random walk simulations indicates that the time course of amperometric spikes measured near the cell surface is due to a low apparent diffusion coefficient of catecholamines near the cell surface and not due to slow dissociation from the granular matrix.

There are several hypotheses concerning the pathogenesis of hypertrophic cardiomyopathy (genetic, ischaemic, immune, inflammatory and apoptosis induction). We have studied three types of cardiomyopathy in order to observe the expression and assess the significance of different immunohistochemical markers (muscular actin, CD-31, proliferation cell nuclear antigen -PCNA-, Ki-67, and markers related with programmed cell death, bcl-2, p-53 and apopDETEC). We studied different microscopic (haematoxylin-eosin and Masson's thrichrome) and immunohistochemical parameters (streptavidin-biotin-peroxidase and "in situ" hybridisation) of forty cases: ten each of hypertensive hypertrophic cardiomyopathy, essential hypertrophic cardiomyopathy, hypertrophic cardiomyopathy in patients treated with chemotherapy and morphologically "normal" hearts. Our findings point to an absence of structural marker expression (actin and CD-31) in cases of hypoxic damage. The distribution and intensity of apoptosis markers, a seen by "in situ" hybridisation were irregular, and the rest of the markers studied showed negative results, with the exception of acridin orange (a marker of hypoxic damage). In our opinion, the above immunohistochemical markers, especially actin and CD-31, could be used for differentiating hypoxic lesions in these three types of cardiomyopathy. Moreover, it is difficult to know the significance of the apoptosis markers, because the autolysis process produces cross reactions with false positive results. We think that there is a need for new studies on DNA breakdown processes during the post-mortem interval. To avoid autolysis problems the post-mortem material needs to be as fresh as possible.

The present report was designed to study the macrophage role and their association with percentage of spermatozoa showing DNA native, morphologically normal sperm and occurrence of antibody-coated spermatozoa. Human sperm samples from 31 patients were used in this study. All samples were evaluated for standard semen parameters according to World Health Organization criteria. The sperm heads with abnormal versus normal chromatin structure were specified as orange-red versus green by Acridine Orange method. Macrophage concentration was assessed with Neutral Red in Neubauer hemocytometer. The technique for the determination of sperm bound antisperm antibodies was sperm mixed antiglobulin reaction. In our study the presence of antisperm antibodies in associated to macrophages concentration (p < 0.05). The log of phagocytic cells were highly correlated with an increase of spermatozoa showing DNA denaturation (p < 0.05). The log of phagocytic cells were highly correlated with an increase of spermatozoa showing DNA denaturation (p < 0.05). There isn't significative association between the log macrophage concentration and morphologically normal sperm (p: 0.414). Thus, the present study further emphasizes the importance of immunosurveillance of the macrophages, and the contribution at the semen quality for the success of the fertilization.

Severe neutropenia following cytotoxic, anti-cancer chemotherapy is well-known to be associated with an increased risk of infections that may be life-threatening, particularly if not treated immediately. Consequently, serial measurements of neutrophil counts in peripheral blood are done routinely following the administration of high-dose myelosuppressive chemotherapy in order to monitor the onset, severity, and duration of iatrogenic neutropenia. We have studied a non-invasive method of quantifying neutrophils recoverable from the oral mucosa, a normal tissue site of neutrophil turnover, as an alternative approach for monitoring severe, chemotherapy-induced neutropenia. This method is based on the quantification of fluorochrome-stained neutrophils present in timed mouthwash specimens. Blood neutrophil (ANC) and mucosal neutrophil counts (MNC) were measured repeatedly in 23 patients who had been treated with dose-intensive chemotherapy for a variety of indications. All 23 patients developed profound neutropenia (ANC < 100/mm3), and 19 developed neutropenic fever (>101 degrees F) during the 2 weeks following treatment. Nadirs of neutropenia defined by MNC were significantly less prolonged than those defined by the ANC. Furthermore, the onset and resolution of neutropenic fever coincided more precisely with nadirs of neutropenia defined by the MNC than with those defined by the ANC. Our findings indicate that oral mucosal neutrophil counts predict the timing of clinical events associated with neutropenia (e.g., the onset and resolution of fever) with significantly greater accuracy than blood neutrophil counts., (Copyright 2002 Wiley-Liss, Inc.)

T cell proliferative responses decrease with age, but the mechanisms responsible are unknown. We examined the impact of age on memory and naive CD4(+) T cell entry and progression through the cell cycle using acridine orange to identify cell cycle stage. For both subsets, fewer stimulated cells from old donors were able to enter and progress through the first cell cycle, with an increased number of cells arrested in G(0) and fewer cells in post G(0) phases. The number of dead cells as assessed by sub-G(0) DNA was also significantly greater in the old group. CD4(+) T cells from old mice also exhibited a significant reduction in clonal history as assessed by CFSE staining. This was associated with a significant decline in cyclin D2 mRNA and protein. We propose that decreases in cyclin D2 are at least partially responsible for the proliferative decline found in aged CD4(+) T cells.

This unit provides a detailed method for evaluating in situ DNA denaturation by flow cytometry. The principal technique is based upon the metachromatic properties of acridine orange. This technique has a number of advantages over traditional biochemical methods, but requires very precise methodology; the result is excellent clarity and differentiation between single-stranded and double-stranded DNA. This unit provides full details on the correct use of acridine orange together with an excellent discussion of the pitfalls and problems.

The classical model of secretory vesicle recycling after exocytosis involves the retrieval of membrane (the omega figure) at a different site. An alternative model involves secretory vesicles transiently fusing with the plasma membrane (the 'kiss and run' mechanism) [1,2]. No continuous observation of the fate of a single secretory vesicle after exocytosis has been made to date. To study the dynamics of fusion immediately following exocytosis of insulin-containing vesicles, enhanced green fluorescent protein (EGFP) fused to the vesicle membrane protein phogrin [3] was delivered to the secretory vesicle membrane of INS-1 beta-cells using an adenoviral vector. The behaviour of the vesicle membrane during single exocytotic events was then examined using evanescent wave microscopy [4-6]. In unstimulated cells, secretory vesicles showed only slow Brownian movement. After a depolarizing pulse, most vesicles showed a small decrease in phogrin-EGFP fluorescence, and some moved laterally over the plasma membrane for approximately 1 microm. In contrast, secretory vesicles loaded with acridine orange all showed a transient (33-100 ms) increase in fluorescence intensity followed by rapid disappearance. Simultaneous observations of phogrin-EGFP and acridine orange indicated that the decrease in EGFP fluorescence occurred at the time of the acridine orange release, and that the lateral movement of EGFP-expressing vesicles occurred after this. Post-exocytotic retrieval of the vesicle membrane in INS-1 cells is thus slow, and can involve the movement of empty vesicles under the plasma membrane ('kiss and glide').

Background: A reliable, validated method for rapidly determining HPC viability is essential for clinical cell engineering., Study Design and Methods: A fluorometric cell viability assay using acridine orange and propidium iodide (AO/PI) was compared to the current standard, trypan blue (TB) exclusion. Viable cells stained with AO/PI fluoresce green under darkfield fluorescence microscopy, while nonviable cells fluoresce orange. Mixtures of fresh and heat-killed bone marrow were prepared and used as viability standards for evaluation of both assays. The frequency of CFU-GM was determined for each specimen., Results: Cell viability measured by AO/PI was extremely linear, with measured and predicted viability in agreement from 0 to 100 percent of the viable cells and a coefficient of regression (r(2)) of 0.9921. The predicted-viability regression line fell within the 95% CI for AO/PI-measured viability. The coefficient of regression for TB-measured viability was 0.9584, with the predicted-viability regression line almost entirely outside the 95% CI. TB overestimated the percentage of viable cells, particularly below the 50-percent level. CFU-GM frequency correlated better with cell viability measured by AO/PI (r(2) = 0.979) than with that measured by TB (r(2) = 0.930)., Conclusions: The AO/PI viability assay is a rapid, highly linear, functionally correlated assay that is superior to conventional viability measurement by TB exclusion.

Vertebrate mechanosensory hair cells contain a narrow "pericuticular" zone which is densely populated with small vesicles between the cuticular plate and cellular junctions near the apical surface. The presence of many cytoplasmic vesicles suggests that the apical surface of hair cells has a high turnover rate. The significance of intense membrane trafficking at the apical surface is not known. Using a marker of endocytosis, the styryl dye FM1-43, this report shows that rapid apical endocytosis in zebrafish lateral line sensory hair cells is calcium and calmodulin dependent and is partially blocked by the presence of amiloride and dihydrostreptomycin, known inhibitors of mechanotransduction channels. As seen in lateral line hair cells, sensory hair cells within the larval otic capsule also exhibit rapid apical endocytosis. Defects in internalization of the dye in both lateral line and inner ear hair cells were found in five zebrafish auditory/vestibular mutants: sputnik, mariner, orbiter, mercury, and skylab. In addition, lateral line hair cells in these mutants were not sensitive to prolonged exposure to streptomycin, which is toxic to hair cells. The presence of endocytic defects in the majority of zebrafish mechanosensory mutants points to a important role of apical endocytosis in hair cell function., (Copyright 1999 John Wiley & Sons, Inc.)

Keloids are collagenous lesions acquired as a result of abnormal wound heating. In this study we have assessed the potential role of proliferation, apoptosis, and necrosis in keloids. Samples were immunolabeled for proliferating cell nuclear antigen or DNA strand breaks or stained with acridine orange. Proliferating cells were observed in the basal layer of the epidermis and fibroblasts in the dermis, the numbers of the latter being increased in comparison with normal skin. No proliferating cells were observed in the central region of the keloid. In normal skin, apoptotic cells were restricted to the basal layer of the epidermis. In keloid samples, numerous apoptotic cells were observed in the epidermis and dermis; the number and distribution of positive cells decreased more distal to the keloid lesion. Apoptotic endothelial cells of a small proportion of blood vessels in the dermis were also observed. Evidence of necrosis was also seen in the dermis. These results suggest that, with maturity, progressive cell degeneration primarily by apoptosis results in clearance of certain cellular populations resulting in the typical keloid lesion. However, the persistence of fibroblast proliferation at the dermal/keloid interface propagates the fibrosis.

V79 cells incubated with di- or tetrasulfonated aluminium phthalocyanines (AlPcS2 or AlPcS4) showed a granular fluorescence pattern. Co-staining with the lysosomotropic dye acridine orange (AO) indicated that the granules that were stained by these photoactive phthalocyanines were identical to lysosomes. Small light exposures made the lysosomes permeable to the dyes without inactivating the cells. Also, the lysosomal enzymes beta-AGA and cathepsin (L+B) were inactivated by small light exposures when AlPcS4 was present. Such small and almost nontoxic light exposures caused a redistribution of the dyes in the cells that was accompanied by a more than 10-fold increase in the fluorescence quantum yields of the dyes. Surprisingly, this redistribution and increase in fluorescence did not result in any significant increase in the photosensitivity of the cells.

The purpose of the present study was to examine the distribution pattern of acridine orange (AO) chromatin interaction products (AOCI) in human neuroblastoma IMR-32 cells and to test whether AO labeling is correlated with BrdU incorporation, and immunohistochemical localization of DNA polymerase alpha, and human N-myc-gene product. Effects of aphidicolin, alpha-amanitin, and actinomycin D on visualization of AO binding to euchromatin and on N-myc-gene expression were also examined. About 25% of the cell nuclei in logarithmic growth phase were immunohistochemically demonstrated to be labeled with BrdU after incubation at 37 degrees for 30 min, indicating cells in DNA synthesis. Most of the cell nuclei showed positive immunoreactivity to DNA polymerase alpha, while human N-myc gene product was found in about 60-80% of the cell nuclei. Electron microscopic studies revealed that about 25% of neuroblastoma cells showed characteristic AOCI within cell nuclei. In the presence of aphidicolin, alpha-amanitin, and actinomycin D, positive cells for N-myc gene product decreased markedly. Percentages of AO positive cells and numbers of AOCI per cell nucleus also showed a marked decrease. But northern blot analysis demonstrated that the expression level of N-myc gene was only repressed by the transcriptional inhibitors alpha-amanitin and actinomycin D. However, no repression was caused by aphidicolin. The present and previous studies of the authors suggest that the ultracytochemical AO method may be indicative for conformational changes of chromatin of cells confined to the cell cycle. Inhibitors of RNA and DNA synthesis then may change the conformational state of chromatin.(ABSTRACT TRUNCATED AT 250 WORDS)

We analyzed commercial samples of acridine orange using reverse-phase high-performance liquid chromatography. The mobile phase of 90:10:2.5 acetonitrile:de-ionized water:pentane sulfonic acid gave baseline separations of components of acridine orange samples in 15 min. Many of the samples were fairly homogeneous; the absorbance due to the acridine orange component ranged from 83-97%. Among the 11 samples tested, one was not acridine orange, reputedly different samples were identical, and the principal component of one sample was not acridine orange.