Simultaneous staining of ribonucleic and deoxyribonucleic acids in unfixed cells using acridine orange in a flow cytofluorometric system. However, currently available methods for quantifying apoptosis have various limitations including multiple, sometimes cell damaging steps, the inability to quantify live, necrotic and apoptotic cells at the same time, and nonspecific detection i. Ao interacts with dna and rna by intercalation or electrostatic attraction respectively. We evaluated the effect of varicocelectomy on semen parameters and levels of sperm dna damage in infertile men. The zebrafish as a model organism for the study of. However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across. Recently it was shown that ao can also be used in live imaging to observe cell death in the zebrafish embryonic spinal cord in a model of inherited cognitive disease related to mutant tau.
Acridine orange is a cell permeable fluorescent dye that binds to nucleic acids, resulting in an altered spectral emission. Acridine orange hydrochloride solution has been used to study autophagic cell death. The vital dye acridine orange was used to identify apoptotic cells. Acridine orange ultra pure enz52405 enzo life sciences. However, zebrafish do have a number of compelling inherent advantages including. Wash 3 times during 10 minutes shacking is optional. Sigma2 receptor ligands have been studied for treatment of pancreatic cancer because they are preferentially internalized by proliferating cells and induce apoptosis. In contrast, antiapoptotic drugs decrease cell death. For teratotoxicity assay, after zebrafish embryos exposed to scde for 24 and 48 hpf, nonlethal malformations were observed under the microscope leica, leica microsystems, bannockburn, il.
Acridine orange staining solution thermo fisher scientific. Wntdkk negative feedback regulates sensory organ size in. The stained cells fluoresce orange when exposed to near ultraviolet light and the method is particularly useful for estimating the total number of microorganisms in samples, such as soil and water, where the simultaneous existence of metabolically diverse populations are encountered. At the 4cell stage approximately one hrpostinjection, hpi, embryos were analyzed by immunofluorescence to detect activated caspase 3. Visualization of dead cells with acridine orange protocols. Caspasemediated apoptosis induction in zebrafish cerebellar. Developmental apoptosis mediates entry and positioning of. Zebrafish embryos are well suited for cell biological and. The cell suspensions were then centrifuged at g for 7 min at 4 c. Amarantemendes, deborah finucane, thomas brunner, ella bossywetzel, and douglas r.
Im using zebrafish as invivo models to study apoptosis induced by metalcompounds. In a subsequent publication, we will examine the question of the inability of pre. Frontiers neuroprotective role of the pi3 kinaseakt. Unique spectral signatures of the nucleic acid dye acridine orange can distinguish cell death. Acridine orange staining did not reveal apoptotic cells in neuromasts, however figure s3d. How does acridine orange works to recognize apoptosis. It is cell permeable, and interacts with dna and rna by intercalation or electrostatic attractions. Histological methods protocols zfin community wiki. Acridine orange is a metachromatic dye which can stain dna, rna and acid glycosaminoglycans. Rna electrostatically bound ao fluoresces red 630nm. Acridine orange, 10 mgml in water high purity ao biotium. In addition, the differential gene expression of ap treated zebrafish embryos was examined by rtpcr analysis.
Significantly more tunelpositive and acridine orange labeled dying cells were found in gef mutant embryos at 2 dpf relative to wildtype embryos. Using the zebrafish danio retio model, we asked whether apoptosis or autophagy was a default. Aug 21, 20 the toxicity study of silver nanoparticles in zebra fish embryos revealed that the acridine orange staining to study apoptosis showed no significant staining in control embryos whereas the agbsa and agstarch treated embryos showed green fluorescent spots on the body, which could be explained using the decomposition of body parts. Interrelationships of acridine orange particles and cytoplasmic reddening prelysosomal acidic vacuoles in dictyostelium discoideum. In the past 5 years, a detailed picture has begun to emerge of the molecular. This cell permeant cellular stain can be utilized in conjunction with a number of other staining techniques and fluorogenic substrates including the. This unique characteristic makes acridine orange useful for cell cycle studies. Unique spectral signatures of the nucleic acid dye acridine orange can distinguish cell death by apoptosis and necroptosis. Acridine orange staining and visualization in zebrafish to. Analyzing apoptosis has been an integral component of many biological studies. Dynamics of acridine orangecell interaction journal.
Image stacks of acridine orange stained embryos treated with 0 a,d, 0. Acridine orange staining is a sensitive, rapid and reliable method for detecting bacteria in blood cultures early during incubation and can be substituted for blind subcultures. Being cell permeable, it interacts with dna and rna by intercalation or electrostatic attractions respectively. We offer a highly purified form of acridine orange while most of the. Incubate alive embryos at room temperature with acridine orange 1x during 30 minutes in e3 medium. We report the design, synthesis and application of several new fluorescent probes lysoprobes ivi that facilitate lysosomal ph monitoring and. In this study we use acridine orange ao staining combined with gen5 microplate reader and imager oftware to determine the amount of apoptotic cell death in zebrafish embryos after 24 hours of ethanol exposure. It has also been used for the staining of chromosomes. Then, the cells were washed with pbs 3%fbs for 2 times. A quick and easy method to determine the number of viable, apoptotic and necrotic cells in a culture.
Acridine orange propidium iodide double staining staining. Cell apoptosis acridine orange detection kit kit genscript. A diagram showing the principle of fluorescence from pierce homepage. Automated fluorescence cell counting application note fluorescence cell counting fluorescence dyes for cell counting fig 2. We evaluated multiple sigma2 receptor ligands in this study, each shown to decrease tumor burden in preclinical models of. Our data show that following zvad treatment, the number of acridine orange positive cells increased in both control figure 4 a,h and scn1labdepleted larvae figure 4 b,i, suggesting that zvad had no effect on cell apoptosis in scn1lab model. Tunel fluorescent labelling and acridine orange staining of the morphants showed high rates of cell death in the head and tail regions. Semen analysis and sperm dna damage expressed as the dna fragmentation index using acridine orange staining and chromatin condensation test by flow cytometry were assessed before. Moreover, the number of hair cells did not change significantly over time in dkk2 overexpressing embryos figure 2 a, suggesting that dkk2 overexpression does not lead to substantial cell death in neuromasts. Acridine orange has also been used as a lysosomal dye. Being cellpermeable, it interacts with dna and rna by intercalation or electrostatic attractions respectively. To assess cell death as a possible mechanism, we analyzed apoptotic cells in embryos by acridine orange staining and tunel assay.
It is used as a nucleic acidselective fluorescent cationic dye useful for cell cycle determination. The multichamber a8slide figure 2 enables high speed viability and cell count determinations of insect and mammalian cells. The levels of apoptosis in irradiated zebrafish embryos at 24hpf were quantified through staining with the vital dye acridine orange, followed by counting the stained cells under a florescent. Thus by many criteria cell death in zebrafish is apoptotic. Neuroprotective role of the pi3 kinaseakt signaling pathway in zebrafish shuang chen, yunzhang liu, xiaozhi rong, yun li, jianfeng zhou and ling lu key laboratory of marine drugs ocean university of china, chinese ministry of education, school of medicine and pharmacy, ocean university of china, qingdao, shandong, china. Acridine orange staining of the mammalian fibroblast cell coat. We perform image processing and analysis to generate a focused image from an image stack and then use the object masking capabilities of gen5 to count the number of green ao positive cells. The cell suspension was triturated by pipetting with a narrow tip at room temperature with continual checks under a dissection microscope for complete digestion. Zebrafish dou yan mutation causes patterning defects and. Inappropriate cell death creates abnormal patterns of apoptosis.
Developmental toxicity and antiinflammatory effect of the. Because of its weak basic property, it accumulates in lysosomes, which have a low ph inside, due to an atpdependent proton pump, present in their membrane. Acridine orange staining solution for use with fluorescent microscopy. Several challenges have limited more widespread use of zebrafish as the organism of choice for drug screening.
Apr 07, 2010 acridine orange ao is a vital dye often used as a marker of apoptotic cells in zebrafish. Acridine orange definition of acridine orange by medical. A d side views of 6dayold living zebrafish larvae expressing taudsred a, see single acridine orange positive neuron in inset, depicted by arrowhead, only dsred b, or no transgene siblings, c stained with acridine orange. In order to assess the levels of cell death after injury we used the acridine orange live staining as described by others 67. The cell suspension is previously mixed with the fluorophores acridine orange and dapi to stain the total cell population and the dead cells, respectively. Cell death in zebrafish embryos increases in a dose dependent manner after treatment with ethanol as shown by number of ao positive cells. Early developmental pathology due to cytochrome c oxidase. Ao will stain all nucleated cells to generate green fluorescence.
Shown is a comparison of nomarski ac and fluourescence df images of the same embryos, stained with acridine orange to detect apoptotic cell death. Additionally, we selected acridine orange ao and 4s green plus nucleic acid stain 4s green as the probe, which can intercalate into the base pairs of doublestranded dna, and the fluorescence intensity is enhanced, and they offer lower toxicity, higher stability, and convenience of using 27. Treat embryos with tricaine or an equivalent anesthetic. When bound to dna, it is very similar spectrally to fluorescein, with an excitation maximum at 502 nm and an emission maximum at 525 nm green. Although this method may label many of the same apoptotic cells. Acridine orange staining is nice for a quick and dirty test of cell death, but tunel is the gold standard and still easy to do wholemount or on sections.
A simple technique for quantifying apoptosis in 96well. With the nucleic acid binding dyes acridine orange ao and propidium iodide pi you can accurately determine cell viability. Acridine orange apoptosis indicator dye was incorporated into the treatment protocol, and it was observed that irregular epithelial cell death was occurring in treated embryos but not in the control group. Lysosomal membrane permeabilization is an early event in. Shop online for a wide selection of bd acridine orange stain for detecting microorganisms in direct smears by fluorescence. With cell dna and rnabinding capacity gaps exist, it may issue a different color fluorescence, and dna binding of less green fluorescence, and the amount of rnabinding orange or orange red fluorescence. Acridine orange staining and visualization in zebrafish to study. Protocol acridine orange ethidium bromide aoeb staining to detect apoptosis. Im using zebrafish as invivo models to study apoptosis induced by metal compounds. The plugnplay assays include 5 different apoptosis assays, a 2step cell cycle analysis and gfptransfection assay in addition to a platform for userdefinable assays. Analysis of apoptosis in live embryos with acridine ora. When ao intercalates into dsdna it emits green fluorescence upon excitation at 480490 nm. Use of zebrafish apoptosis assays for preclinical drug. Immunostaining with the antiphosphohistone h3 ph3 antibody suggested that the abnormalities of ptmab morphants could be due to defective cell proliferation that results in growth imbalances.
The phenotype of the good effort mutant zebrafish is. Differential staining of dna and rna in intact cells and isolated cell nuclei with acridine orange. Developmental toxicity and alteration of gene expression in. We have worked out a cytochemical method, which is suitable for the specific demonstration of surface gag components.
Advantages of acridine orange staining include the speed and ease of the staining. Acridine orange staining solution for lysosome and dna. After 2h, 1ugml acridine orange was added directly to the medium and incubated for 20min at 37c. The wholemount analysis provides spatial information in regard to tissue specificity. Candida albicans under fluorescence microscope stained with acridine orange duration. Acridine orange is a nucleic acid selective fluorescent cationic dye useful for cell cycle determination. Acridine orange is the most widely used fluorescent dye for counting the number of both living and dead bacterial cells. Acridine orange is a fluorescent dye which easily traverses the cell membrane. Acridine orange is a fluorescent pigment, the detection wavelength of 488 nm excitation filter. Sternheimermalbin staining solution for use with light microscopy.
This has especially been applied i to cytodiagnosis of cancer. When bound to dna, it is very similar spectrally to fluorescein, with an excitation maximum at 502 nm and an emission maximum at 525 nm. The overall goal of this method is to identify the relative abundance and location of apoptotic cells in zebrafish from the 4 cell stage to 32 hrpostfertilization hpf. Using the vital dye acridine orange to detect dying cells. Cell death was assessed using acridine orange staining combined with realtime confocal imaging. 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. Sigma2 receptor ligandmediated cell death is dependent on lysosomal accumulation and membrane permeabilization. Ssulfocysteine induces seizurelike behaviors in zebrafish. Acridine orange propidium iodide double staining for apoptotic and necrotic cell determination.
Acridine orangeethidium bromide aoeb staining to detect. Live dead cell imaging kit on the evos auto imaging system duration. Recently it was shown that ao can also be used in live imaging to observe cell death in the zebrafish embryonic spinal cord in a model of inherited cognitive disease related to mutant tau 34. A member of the class of aminoacridines that is acridine carrying two dimethylamino substituents at positions 3 and 6. The nucleocounter instruments are designed for the dedicated cell biology research and development laboratories. A zebrafish model of tauopathy allows in vivo imaging of. In this first publication, we provoke cell death in zebrafish embryos and evaluate the form of cell death by several means, to demonstrate that this death is apoptotic by all usual criteria. Oct embedding for cryostat sectioning of embryos or larvae. When cell death was examined using an acridine orange staining method, a dramatic change in the number of neuronal cell deaths in embryos was observed, especially in the cns of embryos that overexpress gm3 synthase.
At low concentration it intercalates into dna and precipitates rna. Acridine orange is better than gram stain in cases with low amounts of organisms. Vectacell acridine orange for live cell imaging of organelles. Washingdilution buffer 20 x concentrated phosphate buffered saline pbs with 2% bovine serum albumin and 2% sodium azide. Neural crest survival and differentiation in zebrafish. Analysis of apoptosis in zebrafish embryos by wholemount immunofluorescence to detect activated caspase 3. Using the vital dye acridine orange to detect dying cells in. A rapid apoptosis assay measuring relative acridine orange fluorescence in zebrafish embryos. Acridine orange staining has been shown to be highly selective for apoptotic cells in drosophila. Spectral properties of acridine orange bound to dnarna mo jiang abstract dnarna, present in almost every cell, has been a hot research topic in a wide range of areas, including biology, physics, chemistry and polymer engineering. Turck staining solution for use with light microscopy. In vivo imaging of neuronal cell death in tauexpressing zebrafish. Naturally, to image dna and facilitate dnarnarelated questions becomes an interesting question itself.
Acridine orange staining of living embryos revealed increased staining of brain cells especially the hindbrain region of nestin mo treated embryos at 29 hpf fig. Zebrafish as an in vivo screen for early black cranberry. Dec 20, 20 wholemount immunofluorescence to detect activated caspase 3 casp3 assay is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. Using gen5 to analyze cell death in zebrafish embryos. Cell death was detected in live embryos using acridine orange staining at 72 hpf kang et al. Protocol for acridine orange staining after dna denaturation acridine orange ao is a metachromatic dye which differentially stains doublestranded ds and singlestranded ss nucleic acids. For tunel analysis, embryos were staged and fixed as for in situ hybridization and stored in methanol. When acridine orange associates with rna, the excitation maximum shifts to 460 nm, and the emission maximum sh. The zebrafish danio rerio is a powerful vertebrate model organism that has been extensively used to study apoptotic cell death during normal development and under conditions of cellular stress. Ganglioside gm3 is involved in neuronal cell death the.
The effects of astragalus polysaccharide on zebrafish cell. A accumulation of sigma2 receptor ligands sw120 and pb385 in bxpc3 cells following inhibition of lysosomal ph gradient with the vatpase inhibitor concanamycin a cma 10 nm detected by flow cytometry. Acridine orange ao exhibits metachromatic fluorescence that is sensitive to dna conformation, making it useful for detecting apoptotic cells. Using acridine orange to measure cell death in ethanol.
The size of an organ is largely determined by the number of cells it contains, which in turn is regulated by two opposing processes, cell proliferation and cell death, however, it is generally not cl. Because this time was earlier than any observable gross morphological differences, this cell death was likely the cause of the gross morphological defects. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, casp3 and tunel assays take considerably longer to complete 24 days. Acridine orange is used for fluorescence microscopy and flow cytometry analysis of cellular physiology and cell cycle status. Ectopic neuronal cell death and nucleotide signaling affect microglial cell number. Toxicity effect of silver nanoparticles in brine shrimp artemia. Agar embedding for cryostat sectioning of embryos or larvae. Cell death apoptosis acridine orange zebrafish introduction ethanol exposure from maternal consumption of alcoholic beverages has been linked to developmental abnormalities in both humans and animal models. Acridine orange as a cytotopochemical reagent for nucleic acids dna and rna is discussed in detail. This cell permeant cellular stain can be utilized in conjunction with a number of other staining techniques and fluorogenic substrates including the magic red line of fluorogenic protease substrates. Role of programmed cell death in defining zebrafish development. Analysis of apoptosis in zebrafish embryos by wholemount.
Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, casp3 and tunel assays take. In response to proapoptotic agents, cell death increases in zebrafish embryos. At low ph inside the organelles, it will emit an orange fluorescence peak at 590 nm. Using acridine orange to measure cell death in ethanol treated. Spectra of acridine orange and propidium iodide from invitrogen homepage. In the present study, we used a combination of behavior analysis and vital dye staining to show that ssc induces increased swimming, seizurelike movements, and increased cell death in the central nervous system of zebrafish larvae. These results strongly suggest that the overexpression of gm3 causes neuronal cell death in the in vivo zebrafish animal model system. Acridine orange sigma a6014 is prepared at 1mgml 100x in milliq water and stored at 20c light protected. Unique spectral signatures of the nucleic acid dye. The dye is membranepermeant and its nucleic acid binding property has been used for cell cycle studies.
The hydrochloride salt is the fluorescent dye acridine orange, used for cell cy cle determination. Principle, procedure, results and applications august, 2015 nisha rijal staining techniques in microbiology 1 acridine orange is a dye that intercalates or binds with the nucleic acid either dna or rna present in organisms and fluoresce to emit various colors that help in differentiation of cellular organells. Various concentrations of proanthocyanidins in solution were tested against fish ranging in age from 1 cell stage to adult level of growth. B,e homozygous mutant noi tu29a embryo at 22 h, stained with acridine orange. Apoptosis plays important roles in embryogenesis, tissue homeostasis, and immune system regulation. A total of 75 infertile men with varicocele and 40 fertile men controls were included in this study. Highly stable and sensitive fluorescent probes lysoprobes. Apr 05, 2015 zebrafish embryo acridine orange kalishwara lal. Centriole amplification in zebrafish affects proliferation and survival but not differentiation of neural progenitor cells. A rapid apoptosis assay measuring relative acridine orange.
Acridine orange is a cell permeant nucleic acid binding dye that emits green fluorescence when bound to dsdna and red fluorescence when bound to ssdna or rna. The method consists of a glutaraldehyde prefixation, an acridine orange blockstaining for 48 h and an oso4 postfixation for some hours. Tunel terminal transferase mediated dutp nick endlabeling and acridine orange labeling were used to assess apoptosis in mob m610 mutants. Finally, pbs 3%fbs was added to cover the cells and observed the staining under a confocal microscopy. Biochemistry and cell biology canadian science publishing. We have recently established a novel inducible genetic cell. Acridine orange ao stains dsdna green 525 nm and rna or single stranded dna red 650 nm. Visualize dead cells under a dissecting microscope with a green fluorescent filter.
Cells free fulltext defective excitatoryinhibitory. Bim and noxa mrna induce rapid apoptosis in zebrafish embryos immediately following injection which, if embryos are left to develop, leads to death by 2 hpi4. Acridine orange is an intercalating dye that can permeate both live and dead cells. Acridine orange ao is a metachromatic dye which differentially stains doublestranded ds and singlestranded ss nucleic acids. Acridine orange staining procedure acridine orange ao is a nucleic acid selective metachromatic stain useful for cell cycle determination. Acridine orange ao is a vital dye often used as a marker of apoptotic cells in zebrafish. This mechanism of apoptosis is poorly understood, with varying reports of caspase3 dependence.
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