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. Ssulfocysteine induces seizurelike behaviors in zebrafish. Acridine orange ao is a vital dye often used as a marker of apoptotic cells in zebrafish. Acridine orange is a fluorescent pigment, the detection wavelength of 488 nm excitation filter. Vectacell acridine orange is a fluorescent dye that can be used to stain acidic organelles, such as lysosomes, autosomes or yeast vacuoles. Ao interacts with dna and rna by intercalation or electrostatic attraction respectively. 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. Using acridine orange to measure cell death in ethanol.
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 fluorescent dye which easily traverses the cell membrane. Being cellpermeable, it interacts with dna and rna by intercalation or electrostatic attractions respectively. Use of zebrafish apoptosis assays for preclinical drug. Cells free fulltext defective excitatoryinhibitory. 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. Simultaneous staining of ribonucleic and deoxyribonucleic acids in unfixed cells using acridine orange in a flow cytofluorometric system. Using gen5 to analyze cell death in zebrafish embryos. Protocol acridine orange ethidium bromide aoeb staining to detect apoptosis. B,e homozygous mutant noi tu29a embryo at 22 h, stained with acridine orange.
At the 4cell stage approximately one hrpostinjection, hpi, embryos were analyzed by immunofluorescence to detect activated caspase 3. 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. 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. 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 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. Acridine orange as a cytotopochemical reagent for nucleic acids dna and rna is discussed in detail. 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. Washingdilution buffer 20 x concentrated phosphate buffered saline pbs with 2% bovine serum albumin and 2% sodium azide. A rapid apoptosis assay measuring relative acridine orange fluorescence in zebrafish embryos. Finally, pbs 3%fbs was added to cover the cells and observed the staining under a confocal microscopy. Acridine orange hydrochloride solution has been used to study autophagic cell death.
Acridine orange is the most widely used fluorescent dye for counting the number of both living and dead bacterial cells. Differential staining of dna and rna in intact cells and isolated cell nuclei with acridine orange. We have recently established a novel inducible genetic cell. Acridine orange propidium iodide double staining for apoptotic and necrotic cell determination. 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 vital dye acridine orange was used to identify apoptotic cells. Acridine orange is a cell permeable fluorescent dye that binds to nucleic acids, resulting in an altered spectral emission. A member of the class of aminoacridines that is acridine carrying two dimethylamino substituents at positions 3 and 6. Oct embedding for cryostat sectioning of embryos or larvae. The plugnplay assays include 5 different apoptosis assays, a 2step cell cycle analysis and gfptransfection assay in addition to a platform for userdefinable assays. Wntdkk negative feedback regulates sensory organ size in.
Shop online for a wide selection of bd acridine orange stain for detecting microorganisms in direct smears by fluorescence. Acridine orange staining and visualization in zebrafish to study. 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. Im using zebrafish as invivo models to study apoptosis induced by metal compounds. The hydrochloride salt is the fluorescent dye acridine orange, used for cell cy cle determination. 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. Acridine orange staining of the mammalian fibroblast cell coat. We have worked out a cytochemical method, which is suitable for the specific demonstration of surface gag components. When acridine orange associates with rna, the excitation maximum shifts to 460 nm, and the emission maximum sh. Acridine orange ao is a metachromatic dye which differentially stains doublestranded ds and singlestranded ss nucleic acids. In a subsequent publication, we will examine the question of the inability of pre. Zebrafish embryos are well suited for cell biological and. The wholemount analysis provides spatial information in regard to tissue specificity.
Unique spectral signatures of the nucleic acid dye acridine orange can distinguish cell death. The cell suspension was triturated by pipetting with a narrow tip at room temperature with continual checks under a dissection microscope for complete digestion. Toxicity effect of silver nanoparticles in brine shrimp artemia. Turck staining solution for use with light microscopy. Acridine orange is an intercalating dye that can permeate both live and dead cells. Vectacell acridine orange for live cell imaging of organelles. Acridine orange staining solution thermo fisher scientific. Acridine orange is used for fluorescence microscopy and flow cytometry analysis of cellular physiology and cell cycle status. Zebrafish dou yan mutation causes patterning defects and. Here we describe how to perform wholemount immunofluorescence in early zebrafish embryos to detect cells with activated caspase 3 referred to hereafter as the casp3 assay. However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across.
Although this method may label many of the same apoptotic cells. Live dead cell imaging kit on the evos auto imaging system duration. A simple technique for quantifying apoptosis in 96well. Naturally, to image dna and facilitate dnarnarelated questions becomes an interesting question itself. Treat embryos with tricaine or an equivalent anesthetic. 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. 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.
Using the vital dye acridine orange to detect dying cells in. Acridine orange is better than gram stain in cases with low amounts of organisms. Dynamics of acridine orangecell interaction journal. 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. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, casp3 and tunel assays take. 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. Acridine orange definition of acridine orange by medical. Sternheimermalbin staining solution for use with light microscopy.
Sigma2 receptor ligands have been studied for treatment of pancreatic cancer because they are preferentially internalized by proliferating cells and induce apoptosis. 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. We evaluated multiple sigma2 receptor ligands in this study, each shown to decrease tumor burden in preclinical models of. Role of programmed cell death in defining zebrafish development. 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. 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. Im using zebrafish as invivo models to study apoptosis induced by metalcompounds. 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. 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. A rapid apoptosis assay measuring relative acridine orange.
Acridine orange ultra pure enz52405 enzo life sciences. Zebrafish as an in vivo screen for early black cranberry. Analysis of apoptosis in zebrafish embryos by wholemount immunofluorescence to detect activated caspase 3. Apr 05, 2015 zebrafish embryo acridine orange kalishwara lal. 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. Frontiers neuroprotective role of the pi3 kinaseakt. We report the design, synthesis and application of several new fluorescent probes lysoprobes ivi that facilitate lysosomal ph monitoring and.
For tunel analysis, embryos were staged and fixed as for in situ hybridization and stored in methanol. Acridine orange staining and visualization in zebrafish to. Acridine orange, 10 mgml in water high purity ao biotium. After 2h, 1ugml acridine orange was added directly to the medium and incubated for 20min at 37c. The effects of astragalus polysaccharide on zebrafish cell.
Interrelationships of acridine orange particles and cytoplasmic reddening prelysosomal acidic vacuoles in dictyostelium discoideum. It has also been used for the staining of chromosomes. Incubate alive embryos at room temperature with acridine orange 1x during 30 minutes in e3 medium. Developmental toxicity and alteration of gene expression in. Acridine orangeethidium bromide aoeb staining to detect. 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. The multichamber a8slide figure 2 enables high speed viability and cell count determinations of insect and mammalian cells. The dye is membranepermeant and its nucleic acid binding property has been used for cell cycle studies. Then, the cells were washed with pbs 3%fbs for 2 times. Using the vital dye acridine orange to detect dying cells.
Sigma2 receptor ligandmediated cell death is dependent on lysosomal accumulation and membrane permeabilization. Early developmental pathology due to cytochrome c oxidase. A zebrafish model of tauopathy allows in vivo imaging of. Ao will stain all nucleated cells to generate green fluorescence. Rna electrostatically bound ao fluoresces red 630nm. Acridine orange ao exhibits metachromatic fluorescence that is sensitive to dna conformation, making it useful for detecting apoptotic cells. This cell permeant cellular stain can be utilized in conjunction with a number of other staining techniques and fluorogenic substrates including the. Candida albicans under fluorescence microscope stained with acridine orange duration. Lysosomal membrane permeabilization is an early event in. Ganglioside gm3 is involved in neuronal cell death the.
Agar embedding for cryostat sectioning of embryos or larvae. Because this time was earlier than any observable gross morphological differences, this cell death was likely the cause of the gross morphological defects. In the past 5 years, a detailed picture has begun to emerge of the molecular. Acridine orange staining solution for lysosome and dna.
Spectra of acridine orange and propidium iodide from invitrogen homepage. 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. Cell apoptosis acridine orange detection kit kit genscript. 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. The cell suspensions were then centrifuged at g for 7 min at 4 c. Unique spectral signatures of the nucleic acid dye. Neural crest survival and differentiation in zebrafish. At low concentration it intercalates into dna and precipitates rna. Acridine orange sigma a6014 is prepared at 1mgml 100x in milliq water and stored at 20c light protected. When ao intercalates into dsdna it emits green fluorescence upon excitation at 480490 nm. 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. Cell death was detected in live embryos using acridine orange staining at 72 hpf kang et al.
The nucleocounter instruments are designed for the dedicated cell biology research and development laboratories. The phenotype of the good effort mutant zebrafish is. How does acridine orange works to recognize apoptosis. However, zebrafish do have a number of compelling inherent advantages including. 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. 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. 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. Advantages of acridine orange staining include the speed and ease of the staining. Unique spectral signatures of the nucleic acid dye acridine orange can distinguish cell death by apoptosis and necroptosis. Apoptosis plays important roles in embryogenesis, tissue homeostasis, and immune system regulation. Visualization of dead cells with acridine orange protocols. Cell death was assessed using acridine orange staining combined with realtime confocal imaging. A quick and easy method to determine the number of viable, apoptotic and necrotic cells in a culture. In contrast, antiapoptotic drugs decrease cell death.
Using acridine orange to measure cell death in ethanol treated. Amarantemendes, deborah finucane, thomas brunner, ella bossywetzel, and douglas r. 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. 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. 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. 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 has also been used as a lysosomal dye. Tunel terminal transferase mediated dutp nick endlabeling and acridine orange labeling were used to assess apoptosis in mob m610 mutants. In response to proapoptotic agents, cell death increases in zebrafish embryos. In addition, the differential gene expression of ap treated zebrafish embryos was examined by rtpcr analysis. Developmental toxicity and antiinflammatory effect of the. Inappropriate cell death creates abnormal patterns of apoptosis. When ao intercalates into dsdna, it releases green fluorescence upon excitation at 480490 nm. 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.
This has especially been applied i to cytodiagnosis of cancer. Various concentrations of proanthocyanidins in solution were tested against fish ranging in age from 1 cell stage to adult level of growth. Caspasemediated apoptosis induction in zebrafish cerebellar. The zebrafish as a model organism for the study of. Purkinje cells pcs in transgenic zebrafish that coexpress the inducible. Visualize dead cells under a dissecting microscope with a green fluorescent filter. Analyzing apoptosis has been an integral component of many biological studies. 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. Tunel fluorescent labelling and acridine orange staining of the morphants showed high rates of cell death in the head and tail regions. Biochemistry and cell biology canadian science publishing. It is used as a nucleic acidselective fluorescent cationic dye useful for cell cycle determination. 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. Centriole amplification in zebrafish affects proliferation and survival but not differentiation of neural progenitor cells. Acridine orange ao stains dsdna green 525 nm and rna or single stranded dna red 650 nm.
Acridine orange staining solution for use with fluorescent microscopy. At low ph inside the organelles, it will emit an orange fluorescence peak at 590 nm. Wash 3 times during 10 minutes shacking is optional. A total of 75 infertile men with varicocele and 40 fertile men controls were included in this study.
Ectopic neuronal cell death and nucleotide signaling affect microglial cell number. In vivo imaging of neuronal cell death in tauexpressing zebrafish. Cell death in zebrafish embryos increases in a dose dependent manner after treatment with ethanol as shown by number of ao positive cells. Acridine orange propidium iodide double staining staining. This unique characteristic makes acridine orange useful for cell cycle studies. Several challenges have limited more widespread use of zebrafish as the organism of choice for drug screening. A diagram showing the principle of fluorescence from pierce homepage. Acridine orange staining has been shown to be highly selective for apoptotic cells in drosophila. This mechanism of apoptosis is poorly understood, with varying reports of caspase3 dependence. To assess cell death as a possible mechanism, we analyzed apoptotic cells in embryos by acridine orange staining and tunel assay. Apr 07, 2010 acridine orange ao is a vital dye often used as a marker of apoptotic cells in zebrafish. Being cell permeable, it interacts with dna and rna by intercalation or electrostatic attractions respectively. Acridine orange is a metachromatic dye which can stain dna, rna and acid glycosaminoglycans.
Analysis of apoptosis in zebrafish embryos by wholemount. Histological methods protocols zfin community wiki. Using the zebrafish danio retio model, we asked whether apoptosis or autophagy was a default. Image stacks of acridine orange stained embryos treated with 0 a,d, 0. We offer a highly purified form of acridine orange while most of the. Automated fluorescence cell counting application note fluorescence cell counting fluorescence dyes for cell counting fig 2.
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. 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. With the nucleic acid binding dyes acridine orange ao and propidium iodide pi you can accurately determine cell viability. Acridine orange staining did not reveal apoptotic cells in neuromasts, however figure s3d. Highly stable and sensitive fluorescent probes lysoprobes. Analysis of apoptosis in live embryos with acridine ora. 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. The method consists of a glutaraldehyde prefixation, an acridine orange blockstaining for 48 h and an oso4 postfixation for some hours.
Acridine orange staining procedure acridine orange ao is a nucleic acid selective metachromatic stain useful for cell cycle determination. Thus by many criteria cell death in zebrafish is apoptotic. 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. 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. We evaluated the effect of varicocelectomy on semen parameters and levels of sperm dna damage in infertile men. Developmental apoptosis mediates entry and positioning of. In order to assess the levels of cell death after injury we used the acridine orange live staining as described by others 67. It is cell permeable, and interacts with dna and rna by intercalation or electrostatic attractions. The cell suspension is previously mixed with the fluorophores acridine orange and dapi to stain the total cell population and the dead cells, respectively. These results strongly suggest that the overexpression of gm3 causes neuronal cell death in the in vivo zebrafish animal model system.