Acridine orange

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom, Japan, Switzerland, India

Acridine orange is a fluorescent dye used in various laboratory applications. It is primarily used for nucleic acid staining, allowing the visualization and quantification of DNA and RNA in cells and tissues. Acridine orange exhibits different fluorescence properties when bound to double-stranded DNA (green) or single-stranded RNA (red), making it a useful tool for distinguishing between these biomolecules.

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Close spelling variants of this product

Nonyl acridine orange (NAO) (20 citations) 10-N-nonyl acridine orange (6 citations) Nonyl Acridine Orange (NAO, (6 citations) Acridine Orange solution (5 citations) Acridine orange dye (4 citations) 10-N-nonyl-acridine orange (NAO) (A1372), (4 citations) Acridine orange/ethidium bromide (4 citations) DAPI (4′,6-diamidino-2-phenylindole dihydrochloride), acridine orange (AO), (3 citations) Acridine Orange (AO; (3 citations) Acridine orange/propidium iodide (2 citations)

176 protocols using acridine orange

Quantifying Autophagy, Mitochondrial Changes, and Senescence in Cells

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Acridine Orange - After exposure to chemotherapy or radiation, and recovery, cells were stained with 1 μg/ml Acridine Orange (Invitrogen, Cat# A3568) at 37°C for 25 min and then washed with PBS. Cells were observed under an inverted fluorescence microscope and images taken at 20X magnification. For quantification of autophagy, cells were collected with trypsin and washed with PBS and analyzed by flow cytometry.
LysoTracker and MitoTracker - Cells were seeded in 6-well plates on coverslips. After exposure to chemotherapy or radiation, cells were stained with 100 nM LysoTracker Red DND-99 (Invitrogen, L7528) at 37°C for 30 min and then 100 nM MitoTracker Green FM (Invitrogen, M7514) for an additional 30 min. Cells were observed under a fluorescence microscope and images taken at 100X magnification. Overlapping LysoTracker positive and MitoTracker positive organelles were determined by image overlay.
β-Galactosidase - To quantify β-Gal positive senescent cells, after exposure to Doxorubicin, cells were treated with Bafilomycin (100 nM) for 1 h to achieve lysosomal alkalinization and stained with C₁₂FDG (Invitrogen, Cat#D2893) (10 μM) fluorescent β-Galactosidase substrate for 2 h at 37 ^oC. After incubation, cells were collected, washed, suspended in PBS and analyzed by flow cytometry. All experimental procedures were performed with cells protected from light.

Tyutyunyk-Massey L., Sun Y., Dao N., Ngo H., Dammalapati M., Vaidyanathan A., Singh M., Haqqani S., Haueis J., Finnegan R., Xiaoyan D., Kirberger S., Bos P., Bandyopadhyay D., Pomerantz W., Pommier Y., Gewirtz D, & Landry J.W. (2021). Autophagy Dependent Sensitization of Triple Negative Breast Cancer Models to Topoisomerase II Poisons by Inhibition of The Nucleosome Remodeling Factor. Molecular cancer research : MCR, 19(8), 1338-1349.

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Acridine Orange Staining of Zebrafish

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At 3 dpf, live embryos in six-well plates were incubated in Acridine Orange (Invitrogen) staining solution (1 µg Acridine Orange in E₃ medium) in the dark for 30 min with gentle rocking. Embryos were swiftly washed with E₃ medium four times before being anaesthetised with tricaine, and then loaded into plates for imaging as above. Plates were kept in foil to shield from light and imaged promptly after staining.

Lubin A., Otterstrom J., Hoade Y., Bjedov I., Stead E., Whelan M., Gestri G., Paran Y, & Payne E. (2021). A versatile, automated and high-throughput drug screening platform for zebrafish embryos. Biology Open, 10(9), bio058513.

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Live Embryo Apoptosis Imaging

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At 3dpf, live embryos in 6-well plates were incubated in acridine orange (Invitrogen) staining solution (1 µg acridine orange in E3 medium) in the dark for 30 minutes with gentle rocking. Embryos were swiftly washed with E3 medium four times before being anaesthetised with tricaine, and then loaded into plates for imaging as above. Plates were kept in foil to shield from light and imaged promptly after staining.

Lubin A., Otterstrom J., Hoade Y., Bjedov I., Stead E.R., Whelan M., Gestri G., Paran Y., & Payne E. (2020). A versatile automated high-throughput drug screening platform for zebrafish embryos.

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Apoptosis Assay using Ethidium Bromide and Acridine Orange

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The ethidium bromide/acridine orange assay is the colorimetric assay to define the apoptosis based on the reaction to ethidium bromide (50 μg/mL solution, Thermo Fischer, USA) and the acridine orange (20 μg/mL solution, Thermo Fischer, USA) on MDA-MB-231 cell procured from the NCCS, Pune, India. The cells were cultured in the 12 well plates (Biolite-Thermo) in the concentration of the 2 × 105 cells/2 ml and used the cover slips with Poly l-ornithine solution (0.01 % -#A-004-M, Sigma), followed by incubation for 24 h. The cell culture medium used was DMEM- High Glucose media-(Cat No: 2120785, Gibco). The cells were incubated for another 24 h with test and standard drug (Doxorubicin-#D1515, Sigma) and followed with D-PBS (#TL006, Himedia) wash. The well plates were again washed with D-PBS after removing the cover slips and stained with 200 μl solution. These were again washed, mounted on the fluorescence microscope and emission checked with filter cube of Excitation 560/40 nm, emission with 645/75 nm for EtBr. The excitation of 470/40 nm and emission of 525/50 nm for acridine orange were used for evaluation results [28 (link)].

Jayakumar R., Dash M.K., Kumar P., Sharma S., Gulati S., Pandey A., Cholke K., Fatima Z., Trigun S.K, & Joshi N. (2023). Pharmaceutical characterization and exploration of Arkeshwara rasa in MDA-MB-231 cells. Journal of Ayurveda and Integrative Medicine, 15(1), 100823.

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Analyzing Lysosomal Leakage in Macrophages

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BMDM were treated with LPS at 200 ng mL⁻¹ for 3 h or LLOME at 500 nm for 30 min to induce lysosomal leakage in the presence or absence of GABA as appropriate. Lysosomal leakage was detected with acridine orange^[⁵⁰ (link)
^] or LysoTracker red DND‐99 (L7528, Thermo Fisher Scientific, USA) that specifically stains acidic compartments within a cell. In brief, BMDM grown on coverslips were stained with 2 µg mL⁻¹ acridine orange (A1301, Thermo Fisher Scientific, USA) for 20 min or 50 nm LysoTracker red for 30 min at 37 °C in a humidified 5% CO₂/95% air incubator and washed in PBS before the visualization. Loss of acridine orange staining (Ex 488 nm/Em 650 nm) or LysoTracker red (Ex 577 nm/Em 590 nm) was used as an indicator for lysosomal leakage. The images were captured with Andor Dragonfly 505 confocal spinning disk system. Cell nuclei were stained with Hoechst 33 342 (Ex 405 nm, Em 460 nm) (C1022, Beyotime, China). Quantitative analysis was performed with Imaris Viewer x64 9.6.0 software.

Wang J., Zhang H., Yuan H., Chen S., Yu Y., Zhang X., Gao Z., Du H., Li W., Wang Y., Xia P., Wang J, & Song M. (2024). Prophylactic Supplementation with Lactobacillus Reuteri or Its Metabolite GABA Protects Against Acute Ischemic Cardiac Injury. Advanced Science, 11(18), 2307233.

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Assessing Endosomal Acidification with TVB024

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The effect of TVB024 on endosomal acidification was assessed by acridine orange staining for determination of endosomal pH. Briefly, A549 cells were preincubated with TVB024 for 16 h. The culture media were then replaced with HBSS containing 5 μg/ml acridine orange (A1301; Life Technologies) for 15 min at 37°C. The cells were washed three times with HBSS and then incubated with HBSS containing the drug at the same initial concentrations. The culture plates were then immediately assessed for acridine orange staining using a spectrophotometer equipped with a long pass filter set (479/28 nm excitation/>500 nM emission).

Nicholas B., Staples K.J., Moese S., Meldrum E., Ward J., Dennison P., Havelock T., Hinks T.S., Amer K., Woo E., Chamberlain M., Singh N., North M., Pink S., Wilkinson T.M, & Djukanović R. (2015). A Novel Lung Explant Model for the Ex Vivo Study of Efficacy and Mechanisms of Anti-Influenza Drugs. The Journal of Immunology Author Choice, 194(12), 6144-6154.

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Evaluating TVB024's Impact on Endosomal pH

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The effect of TVB024 on endosomal acidification was assessed by acridine orange staining for determination of endosomal pH. Briefly, A549 cells were pre-incubated with TVB024 for 16 h. The culture media were then replaced with HBSS containing 5μg/ml acridine orange (A1301, Life Technologies) for 15 min at 37°C. The cells were washed three times with HBSS and then incubated with HBSS containing the drug at the same initial concentrations. The culture plates were then immediately assessed for acridine orange staining using a spectrophotometer equipped with a long pass filter set (479/28nm excitation / >500nM emission).

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Fluorescence Microscopy of Stained Cells

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After the incubation, the material was stained with the solution of acridine orange (Invitrogen TM) and Rhodamine B (Invitrogen TM) and then analysed under the fluorescence microscope Nikon Eclipse 80i using the UV-2A (EX- 330-380, DM-400, BA-420) and G-2A filter (EX -510-560, DM-575, BA 590).

Dobrzynski M., Pajaczkowska M., Nowicka J., Jaworski A., Kosior P., Szymonowicz M., Kuropka P., Rybak Z., Bogucki Z.A., Filipiak J., Targonska S., Ciupa-Litwa A., Han A, & Wiglusz R.J. (2019). Study of Surface Structure Changes for Selected Ceramics Used in the CAD/CAM System on the Degree of Microbial Colonization, In Vitro Tests. BioMed Research International, 2019, 9130806.

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Acridine Orange Staining for Autophagy

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Formation of acidic vesicular organelles, a morphological characteristic of autophagy, was detected using acridine orange staining. Cells were incubated with acridine orange (Invitrogen) at 1 μg/mL for 15 min. Red (650 nm, stained cytoplasmic vesicles) vs green (510-530 nm, stained nuclei) fluorescence (FL3/FL1) from cells illuminated with blue (488 nm) excitation light was measured with a FACScan flow cytometer (Beckman Coulter, Brea, CA, United States). The data are presented as the fold changes with an arbitrary setting of autophagy in cells without treatment of drug, hyperthermia or radiation.

Yuan G.J., Deng J.J., Cao D.D., Shi L., Chen X., Lei J.J, & Xu X.M. (2017). Autophagic cell death induced by reactive oxygen species is involved in hyperthermic sensitization to ionizing radiation in human hepatocellular carcinoma cells. World Journal of Gastroenterology, 23(30), 5530-5537.

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Autophagy Assessment in Chronic Lymphocytic Leukemia

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A total of 10⁶ CLL cells were either untreated or treated with IACS-010759 for 24 or 48 h, washed and stained with 1 mL of Acridine Orange (10 μg/mL) (Invitrogen, Carlsbad, CA, USA) and analyzed using flow cytometry for autophagy. Changes in Acridine Orange positive population were determined.

Vangapandu H.V., Alston B., Morse J., Ayres M.L., Wierda W.G., Keating M.J., Marszalek J.R, & Gandhi V. (2018). Biological and metabolic effects of IACS-010759, an OxPhos inhibitor, on chronic lymphocytic leukemia cells. Oncotarget, 9(38), 24980-24991.

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