Acridine orange ao is a nucleic acid selective metachromatic stain useful for cell cycle determination. Acridine orange stain for use with application recommended for use in the fluorescent microscopic detection of microorganisms in direct smears prepared from clinical and non clinical materials. Our results indicate that at least two mechanisms are involved. Acridine orange is cellpermeable, which allows the dye to interact with dna by intercalation, or rna via electrostatic attractions. Induction of autophagic cell death in malignant glioma cells.
A total of 75 infertile men with varicocele and 40 fertile men controls. Acridine orange staining procedure acridine orange ao is a nucleic acid selective metachromatic stain useful for cell cycle determination. Ratiometric analysis of acridine orange staining in the study of. Acridine is an organic compound and a nitrogen heterocycle with the formula c h 9 n. Oxidation of cl is accompanied by a release of cytochrome c from the mitochondria into the cytoplasm, a key event in development of apoptosis. Purified haemoglobin fraction was subjected to sdspage and proteins of approximately 14 kda and 30 kda were. Acridine orange is a cellpermeable fluorescent dye that binds to nucleic acids, resulting in an altered spectral emission. Acridine orange staining and quantification by flow cytometry this is the standard protocol used for the preparation of adherent culture cells used for flow cytometric analysis in this paper. Can anyone recommend a good free facs analysing software. Similar to other animals, in zebrafish embryos, programmed cell death maintains the homeostasis of. Flowcytometry assay for apoptosis using fluorophor 10.
A total of 75 infertile men with varicocele and 40 fertile men controls were included in this study. Acridine orange for cell cycle analysis flow cytometry. I use the bd reagents to clean post acridine orange run on our lsrii. Cell viability assay under 3 min with nc3000 chemometec. Resistance to chemotherapy is one of the major challenges in oncology. Acridine orange ao intercalates into dna, which then emits at. Dec 23, 2010 oxidation of cl is accompanied by a release of cytochrome c from the mitochondria into the cytoplasm, a key event in development of apoptosis. Acridine orange hemizinc chloride salt for nucleic acid. This cellpermeant cellular stain can be utilized in conjunction. Flow cytometry analysis of cell cycle and apoptosis cells were trypsinized 0. Acridine dyes are prepared by condensation of 1,3diaminobenzene derivatives.
All nucleic acidcontaining cells will fluoresce orange. 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. When ao intercalates into dsdna it emits green fluorescence upon excitation at 480490 nm. Optimization of acridine orange staining for buffalo sperm, cryopreserved in egg yolk based extender to detect dna fragmentation mushtaq ahmad 1, mehboob ahmed 1,2 and nasim ahmad 1 1 department of theriogenology, faculty of veterinary science, university of veterinary and animal sciences, outfall road, lahore 54000, pakistan. Acridine orange is an organic compound that serves as a nucleic acidselective fluorescent dye with cationic properties useful for cell cycle determination.
The zproject function creates a focused image projected from an image stack. Evaluation of acridine orange, lysotracker red, and. Induction of autophagic cell death in malignant glioma. 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. Using acridine orange to measure cell death in ethanol. Acridine orange, 10 mgml in water high purity ao acridine orange ao stains dsdna green 525 nm and rna or single stranded dna red 650 nm.
Z projection channel gfp 469,525 method focus stacking size of max filter 11 px top slice 12. For use with application recommended for use in the fluorescent microscopic detection of microorganisms in direct smears prepared from clinical and non. The cassette is a disposable plastic unit that contains the fluorescent dye used for analysis. Acridine orange staining solution for lysosome and dna. Naturally, to image dna and facilitate dnarnarelated questions becomes an interesting question itself. The influence that changes in proton distribution have on the fluorescence of acridine orange was examined using negatively charged liposomes. 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. Dualfluorescence viability, using acridine orange ao and propidium iodide pi, is the recommended method for accurate viability analysis of primary cells, such as pbmcs, splenocytes, and stem cells in samples containing debris and unwanted nonnucleated cell types including red blood cells. Acridine orange 10 mgml in water thermo fisher scientific. 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. Cell counting viability reagents nexcelom bioscience. Acridines are substituted derivatives of the parent ring. Flow cytometry analysis of cell cycle and apoptosis cells were trypsinized.
Intracellular distribution of the fluorescent dye nonyl acridine orange responds to the mitochondrial membrane potential. Feb 09, 2015 dual acridine orange ethidium bromide aoeb staining method was repeated 3 times at least. Often this can be overcome with modern computer data analysis software models. The cellometer vision cba by nexcelom is a dual fluorescent image cytometer for 20 l cellbased assays. Optimization of acridine orange staining for buffalo sperm. Acridine orange rgfir to quantify autophagy by flow cytometry. Felipe1,3 and guido lenz1,4, abstract acridine orange is a cellpermeable green fluorophore that can be. Z projection channel gfp 469,525 method focus stacking size of max filter 11 px top slice 12 bottom slice 1 table 1.
Dualfluorescence viability, using acridine orange ao and propidium iodide pi, is the recommended method for accurate viability analysis of primary cells, such as pbmcs. Waiting times evaluated with the protocol using ao were different, allowing not saying that if the waiting time is longer, between 2. Quantification of avos with acridine orange using facs scan. Differential staining of dna and rna in intact cells and isolated cell nuclei with acridine orange. Cellometer vision cba image cytometer nexcelom bioscience. Bafilomycin was added before facs analysis see materials and methods. Flow cytometry and microscopic acridine orange test.
Anova was used to assess the significance of differences within and between sample groups, and the 401 article flow cytometry acridine orange test to assess semen quality ae apedaile et al. This study aimed to validate a laboratory protocol for flow. Statistical analysis statistical analysis was preformed using minitab. Its metachromatic shift to red fluorescence is concentrationdependent and, therefore, acridine orange fluoresces red in avos, such as autolysosomes. Neuroblastoma is the most common extracranial solid tumor in childhood, and the successful. The green 510530 nm and red 650 nm fluorescence emission of 10 4 cells was measured. Optimization of acridine orange staining for buffalo sperm, cryopreserved in egg yolk based extender to detect dna fragmentation mushtaq ahmad 1, mehboob ahmed 1,2 and nasim. Flow cytometry facility 431 newton road 48 eckstein medical research building iowa city, ia 52242. This information is to serve as a guide and may need to be optimized for particular cell type or flow cytometer equipment. Propidium iodide can only enter dead cells with poor membrane intergrity, so it will stain all dead nucleated cells to generate red fluorescence.
Ao interacts with dna and rna by intercalation or electrostatic attraction respectively. On the contrary, it emits red when interacts with ssdna or rna. Analysis can be timeconsuming unless dedicated software is used. This is a set of tools based on open code with r you need to be a bit advanced to use these tools. Acridine orange is a cellpermeant nucleic acid binding dye that emits green fluorescence when bound to dsdna and red fluorescence when bound to ssdna or rna. Acridine orange 3,6dimethylaminoacridine is a nucleic acidselective metachromatic stain useful for cell cycle determination. Cell apoptosis acridine orange detection kit kit genscript. Do you know detailed protocol for acridine orange staining by flow cytometry for autophagy analysis. Acridine orange has also been used as a lysosomal dye. For flow cytometry, the most useful of these is indo1 whose emission wavelength.
Acridine orange staining solution for lysosome and dna staining. Ao will stain all nucleated cells to generate green fluorescence. With cell dna and rnabinding capacity gaps exist, it. Gen5 image analysis software settings for zprojection.
Ratiometric analysis of acridine orange staining in the study. 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 and 6 months after varicocelectomy. At one time acridine dyes were commercially significant, but they are now uncommon because they are not lightfast. Protocol for acridine orange staining after dna denaturation acridine orange ao is a metachromatic dye which differentially stains doublestranded ds and singlestranded ss. Data were analyzed using cellquest pro software bdb. Flow cytometry protocols flow cytometry protocols researchgate. The stability of fluorescent signals, achievable image contrast, and phototoxicity were taken into consideration. Flow cytometry acridine orange test fcmaot fcmaotwas conducted following the protocol for scsa described by evenson and jost, 1994. Ratiometric analysis of acridine orange staining in the. The volume calibrated single use via2cassette is a true allinone solution for optimized cell count. Acridine orange dye has been used to analyze mitochondria and lysosomal content by flow cytometry, characterize multidrug resistance, and measure changes in mitochondrial mass during. The vision cba system combines the simplicity of image cytometry with the power of flow analysis software to offer userfriendly cellbased assays, featuring. The molar extinction coefficient of haemoglobin in x. In situ analysis of apoptosis in aspergillus nidulans with.
Dual acridine orangeethidium bromide aoeb staining method was repeated 3 times at least. Acridine orange is a fluorescent pigment, the detection wavelength of 488 nm excitation filter. Dual aoeb staining to detect apoptosis in osteosarcoma. Acridine orange is a cellpermeant nucleic acid binding dye that emits green. Do you know detailed protocol for acridine orange staining by flow. Using fluorescence microscopy and image analysis, the nucleocounter nc3000 automates the detection of cells. Acridine orange staining has been shown to be highly selective. We evaluated the effect of varicocelectomy on semen parameters and levels of sperm dna damage in infertile men. Hi wenzel, maybe its to late for answering your question. Dual aoeb staining to detect apoptosis in osteosarcoma cells. The three tested tracers exhibit different advantages. Flow cytometry, nucleic acids analysis, rna content, rna and dna analysis. This unique characteristic makes acridine orange useful for cellcycle studies.
I would like to try acridine orange staining for detection of. Using the vital dye acridine orange to detect dying cells. 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. Acridine orange, which is a metachromatic intercalator sensitive to dna conformation, has been widely used to detect apoptosis in wildtype, mutant, and drugtreated zebrafish brand et al. Histone deacetylase 10 promotes autophagymediated cell. Rna electrostatically bound ao fluoresces red 630nm. 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 and 6 months.
Acridine orange is an intercalating dye that can permeate both live and dead cells. Flowcytometry assay for apoptosis using fluorophor 10 n. The via2cassette preloaded with acridine orange and dapi. The nc3000 viability and cell count assays use the cell. The nc3000 viability and cell count assays use the cell stain acridine orange for cell detection, and the nucleic acid stain dapi for detecting nonviable cells. Flow cytometry offers the possibility of rapid enumeration of. Nov 02, 2018 flow cytometric analysis of xenopus laevis and x. This cellpermeant 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. Acridine orange, a cellpermeable metachromatic fluorescent cationic dye that intercalates dna and rna, is used in fluorescence and epiflouresence microscopy.
Acridine orange is used for fluorescence microscopy and flow cytometry analysis of cellular physiology and cell cycle status. Use the product attributes below to configure the comparison table. Acridine orange hemizinc chloride salt sigmaaldrich. The pea15 protein regulates autophagy via activation of jnk. Principle, procedure, results and applications august, 2015 nisha rijal staining techniques in microbiology 1 acridine orange is a dye that intercalates or binds. This makes acridine orange staining a quick, accessible and reliable method to assess the volume of avos, which. Acridine orange stain principle, procedure and result. Flow cytometry reticulocyte counting using acridine orange.
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