ArticlePDF Available

Acridine Orange/Ethidium Bromide (AO/EB) Staining to Detect Apoptosis

Authors:
Shailaja Kasibhatla at Genomics Institute of the Novartis Research Foundation
Shailaja Kasibhatla
Gustavo P. Amarante-Mendes at University of São Paulo
Gustavo P. Amarante-Mendes
Deborah Finucane
Deborah Finucane
Deborah Finucane
  • This person is not on ResearchGate, or hasn't claimed this research yet.
Thomi Brunner at Universität Konstanz
Thomi Brunner
Show all 6 authorsHide
Download full-text PDF
Read full-text
Download citation

Abstract

Shailaja Kasibhatla, Gustavo P. Amarante-Mendes, Deborah Finucane,Thomas Brunner, Ella Bossy-Wetzel and Douglas R. GreenThis protocol was adapted from “Apoptosis Assays,” Chapter 15, in Cells (eds. Spector etal.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1998. Thisthree-volume set is now out of print; however, some of the microscopy methods wererepublished in Basic Methods in Microscopy, by David L. Spector and Robert D.Goldman.
Content uploaded by Gustavo P. Amarante-Mendes
Author content
All content in this area was uploaded by Gustavo P. Amarante-Mendes
Content may be subject to copyright.
Cold Spring Harbor ProtocolsCold Spring Harbor Protocols
cshprotocols.cshlp.org
doi:10.1101/pdb.prot4493doi:10.1101/pdb.prot4493
Cold Spring Harb Protoc 2006. 2006: pdb.prot4493-
ProtocolProtocol
Acridine Orange/Ethidium Bromide (AO/EB)
Staining to Detect Apoptosis
Shailaja KasibhatlaShailaja Kasibhatla, , Gustavo P. Amarante-MendesGustavo P. Amarante-Mendes, , Deborah FinucaneDeborah Finucane,,
Thomas BrunnerThomas Brunner, , Ella Bossy-WetzelElla Bossy-Wetzel and and Douglas R. GreenDouglas R. Green
This protocol was adapted from “Apoptosis Assays,” Chapter 15, in
Cells
(eds. Spector et
al.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1998. This
three-volume set is now out of print; however, some of the microscopy methods were
republished in
Basic Methods in Microscopy
, by David L. Spector and Robert D.
Goldman.
INTRODUCTION
Acridine orange/ethidium bromide (AO/EB) staining is used to visualize nuclear
changes and apoptotic body formation that are characteristic of apoptosis. Cells
are viewed under a fluorescence microscope and counted to quantify apoptosis.
MATERIALS
Reagents
Cell suspension
AO/EB solution
Equipment
Fluorescence microscope with fluorescein filter and 60X objective
Slides and coverslips
METHOD
1. Incubate 25 µl of cell suspension (0.5 × 106 to 2.0 × 106 cells/ml)
with 1 µl of AO/EB solution. Mix gently. Each sample should be mixed
just prior to microscopy and quantification. Samples must be
evaluated immediately.
2. Place 10 µl of cell suspension onto a microscopic slide, cover with a
glass coverslip, and examine at least 300 cells in a fluorescence
microscope using a fluorescein filter and a 60X objective. (Higher or
lower magnification may be desired depending on cell type. Nuclear
morphology should be discernible.)
Acridine orange is a vital dye and will stain both live and dead cells.
Ethidium bromide will stain only cells that have lost membrane
integrity. Live cells will appear uniformly green. Early apoptotic cells
will stain green and contain bright green dots in the nuclei as a
consequence of chromatin condensation and nuclear fragmentation.
Late apoptotic cells will also incorporate ethidium bromide and
therefore stain orange, but, in contrast to necrotic cells, the late
apoptotic cells will show condensed and often fragmented nuclei.
Necrotic cells stain orange, but have a nuclear morphology
resembling that of viable cells, with no condensed chromatin.
... Early apoptotic cells are observed as green. Green cells with normal morphology observed in the cell population are alive, and red-orange cells with normal morphology are necrotic cells (Kasibhatla et al., 2006). ...
... With fluorescence microscopy, AO enables the visualization of normal and apoptotic nuclei of living cells as green. EB allows the normal and apoptotic nuclei of dead cells to appear red (Kasibhatla et al., 2006;Petit et al., 1993). The experiment was repeated 3 times, 500 cells were counted for each concentration in each repetition. ...
Cytotoxic and apoptotic effects of Prunus spinosa fruit extract on HT-29 colon cancer line
Article
Full-text available
  • May 2024
Colon cancer holds the position of the third most common type of cancer and stands as the third leading cause of cancer-related deaths for both men and women. Modern strategies in cancer prevention center around the use of natural compounds, which demonstrate a range of effects, including preventive, inhibitory, and latency-inducing impacts on the progression of cancer. In the present study, aqueous extracts derived from the fruits of Prunus spinosa L. (blackthorn, Rosaceae) are employed to assess their cytotoxic potential against the HT-29 colon cancer cell line. The fruit extract is administered to the HT29 cell line in different concentrations over 24 and 48-hours to evaluate the induction of apoptosis. The MTT cell viability test is employed to quantify the cytotoxic effect, indicating the extent of the impact. Additionally, the EB/AO (ethidium bromide/acridine orange) dual staining method is utilized to gather supplementary information regarding the cytotoxic effects. Observations after 24 hours of exposure showed no significant cytotoxic effect; however, 48-hour exposure revealed IC20, IC50, and IC80 values of 1.27, 173.7, and > 1000 µg/ml, respectively, as determined by MTT analysis. Correspondingly, values of 5.06, 123.8, and > 1000 µg/ml were recorded by the EB/AO dual staining method. Our results show that P. spinosa fruit water extract has an inhibitory effect on the HT-29 cell viability by exerting cytotoxic and apoptotic effects in a concentration-dependent and time-dependent manner. Toxicity studies have shown that MTT and EB/AO support each other and achieve similar results. Further extensive research into the metabolic and functional effects of P. spinosa could illuminate its potential and increase its economic importance in the field of anticancer treatments as a natural drug.
View
... The homogenate was centrifuged at 4000 rpm for 15 min at 4 °C. The homogenates were used for estimation of the following parameters [total oxidant capacity in testicular and ovarian tissues [22] , and the second part of testes and ovaries were used for detection of ttesticular and ovarian Caspase-3 activity [23] and DNA damage in testicular and ovarian tissues [24] . ...
... Detection of DNA damage in testicular and ovarian tissues by electrophoresis [24] . DNA extraction by EZ-10 Column Genomic DNA Minipreps kit, Animal Test principle: The selective adsorption of DNA on a silicabased membrane embedded in an EZ-10 Spin Column is contingent upon the advantageous properties of silica-based DNA purification technology. ...
View
... Te degree of apoptosis induced by CP-MgONPs in Hep2 cancerous cells was evaluated by dual AO/EB fuorescent labelling [38]. Te cultivated Hep2 cells (1 × 10 5 cells/well) were seeded in a 12-well plate and placed in a humid environment at 37°C with a steady supply of CO 2 (5%) for 12 h. ...
Biogenic Synthesis of Photosensitive Magnesium Oxide Nanoparticles Using Citron Waste Peel Extract and Evaluation of Their Antibacterial and Anticarcinogenic Potential
Article
Full-text available
Background. Magnesium oxide nanoparticles (MgONPs) have been fabricated by several approaches, including green chemistry approach due to diverse application and versatile features. Objectives. The current study aimed to prepare a convenient, biocompatible, and economically viable MgONPs using waste citron peel extract (CP-MgONPs) to evaluate their biological applications. Methods. The CP-MgONPs were synthesized by a sustainable approach from extract of waste citron peel both as capping and reducing agents without use of any hazardous material. The physicochemical features of formed CP-MgONPs were determined by sophisticated analytical and microscopic techniques. The biogenic CP-MgONPs were examined for their antibacterial, anticarcinogenic, and photocatalytic attributes. Results. A prominent absorption peak in the UV-Vis spectra at 284 nm was the distinguishing characteristic of the CP-MgONPs. The scanning electron microscopy (SEM) reveals polyhedral morphology of nanoparticles with slight agglomeration of CP-MgONPs. The CP-MgONPs exerted excellent antibacterial potencies against six bacterial strains. The CP-MgONPs displayed significant susceptibility towards E. coli (20.72 ± 0.33 mm) and S. aureus (19.52 ± 0.05 mm) with the highest inhibition zones. The anticancer effect of CP-MgONPs was evaluated against HepG2 (IC50 : 15.3 μg·mL⁻¹) cancer cells and exhibited potential anticancer activity. A prompt inversion of cellular injury manifested as impairment of the integrity of the cell membrane, apoptosis, and oxidative stress was observed in treated cells with CP-MgONPs. The biosynthesized CP-MgONPs also conducted successful photocatalytic potential as much as MgO powder under the UV-light using acid orange 8 (AO-8) dye. The degradation performance of CP-MgONPs showed over 94% photocatalytic degradation efficiency of acid orange 8 (AO-8) dyes within a short time. Conclusions. Outcomes of this research signify that biogenic CP-MgONPs may be advantageous at low concentrations, with positive environmental impacts.
View
... For the Alzheimer's disease model, the cells were treated with Aβ 42 (0.5 µM) [39], Aβ 42 (0.5 µM) + B. monnieri / C. asiatica extract (100 µg/mL), B. monnieri / C. asiatica extract alone (100 µg/mL) for 48 h along with the untreated control cells. After treatment, the cells were stained using acridine orange: ethidium bromide (2 µg/ mL,1:1) dual stains [40] for 20 min at 37 °C in the dark. Then, the excess stain was removed, and the cells were overlaid with 1X PBS. ...
Comparative Metabolomics and Network Pharmacology Analysis Reveal Shared Neuroprotective Mechanisms of Bacopa monnieri (L.) Wettst and Centella asiatica (L.) Urb
Article
Full-text available
  • May 2024
  • MOL NEUROBIOL
Bacopa monnieri (L.) Wettst and Centella asiatica (L.) Urb., two nootropics, are recognized in Indian Ayurvedic texts. Studies have attempted to understand their action as memory enhancers and neuroprotectants, but many molecular aspects remain unknown. We propose that Bacopa monnieri (L.) Wettst and Centella asiatica (L.) Urb. share common neuroprotective mechanisms. Mass spectrometry–based untargeted metabolomics and network pharmacology approach were used to identify potential protein targets for the metabolites from each extract. Phytochemical analyses and cell culture validation studies were also used to assess apoptosis and ROS activity using aqueous extracts prepared from both herbal powders. Further, docking studies were also performed using the LibDock protocol. Untargeted metabolomics and network pharmacology approach unveiled 2751 shared metabolites and 3439 and 2928 non-redundant metabolites from Bacopa monnieri and Centella asiatica extracts, respectively, suggesting a potential common neuroprotective mechanism among these extracts. Protein-target prediction highlighted 92.4% similarity among the proteins interacting with metabolites for these extracts. Among them, kinases mapped to MAPK, mTOR, and PI3K-AKT signaling pathways represented a predominant population. Our results highlight a significant similarity in the metabolome of Bacopa monnieri (L.) Wettst and Centella asiatica (L.) Urb., and their potential protein targets may be attributed to their common neuroprotective functions. Graphical Abstract Workflow employed for phytochemical assays, mass spectrometry-based metabolomics, protein-target identification, cell culture validations, and docking studies. Comparison of Bacopa monnieri and Centella asiatica non-redundant metabolite identification at MS level,as well as at MS/MS level
View
... Apoptosis can be visualized directly by fluorescence microscopy when the cells are stained with acridine orange/ethidium bromide (AO/EB) 71 . AO (green) stains both live and dead cells. ...
Cell-penetrating protein-recognizing polymeric nanoparticles through dynamic covalent chemistry and double imprinting
Article
Full-text available
  • May 2024
Molecular recognition of proteins is key to their biological functions and processes such as protein–protein interactions (PPIs). The large binding interface involved and an often relatively flat binding surface make the development of selective protein-binding materials extremely challenging. A general method is reported in this work to construct protein-binding polymeric nanoparticles from cross-linked surfactant micelles. Preparation involves first dynamic covalent chemistry that encodes signature surface lysines on a protein template. A double molecular imprinting procedure fixes the binding groups on the nanoparticle for these lysine groups, meanwhile creating a binding interface complementary to the protein in size, shape, and distribution of acidic groups on the surface. These water-soluble nanoparticles possess excellent specificities for target proteins and sufficient affinities to inhibit natural PPIs such as those between cytochrome c (Cytc) and cytochrome c oxidase (CcO). With the ability to enter cells through a combination of energy-dependent and -independent pathways, they intervene apoptosis by inhibiting the PPI between Cytc and the apoptotic protease activating factor-1 (APAF1). Generality of the preparation and the excellent molecular recognition of the materials have the potential to make them powerful tools to probe protein functions in vitro and in cellulo.
View
... The effect of EPS-H29 on the HDF cells was visualized and assessed using acridine orange and ethidium bromide fluorescent staining [39]. The HDF cells were seeded in a 24-well plate at a density of 25,000 cells/well and allowed to attach overnight. ...
Burn Wound Healing Abilities of a Uronic Acid Containing Exopolysaccharide Produced by the Marine Bacterium Halomonas malpeensis YU-PRIM-29 T
Article
Full-text available
  • May 2024
  • APPL BIOCHEM BIOTECH
Bacterial exopolysaccharides (EPS) are an emerging class of biopolymers with extensive applications in different fields due to their versatile physico-chemical and biological properties. The role of EPS in healing of different wound types is gaining interest in the tissue engineering sector. Burn is one of the devitalizing injuries that causes greater physical harm and can be fatal. Appropriate treatment modalities have to be followed for faster healing outcomes and to minimize the risk. In this study, a bacterial EPS (EPS-H29) from the marine bacterium Halomonas malpeensis YU-PRIM-29 T was used to treat the burn wound in vivo. The biochemical and structural characterizations of EPS-H29 were carried out using standard methods. In addition, FE-SEM, conformational, rheological, and HP-GPC analyses were carried out. In vitro biocompatibility of EPS-H29 was studied in human dermal fibroblasts (HDFs) and keratinocytes (HaCaT). Scratch assay was used to study the wound healing in vitro. For in vivo evaluation, burn wound (second-degree) was created on Wistar albino rats and treated with EPS-H29 along with appropriate control groups. The total sugar and protein contents of EPS-H29 were 72.0 ± 1.4% and 4.0 ± 0.5%, respectively, with a molecular weight of 5.2 × 10⁵ Da. The lyophilized samples exhibited porous surface features, and in solution, it showed triple helical conformation and shear thickening behavior. In vitro cell-based assays showed biocompatibility of EPS-H29 up to 200 μg/mL concentration. At a concentration up to 50 μg/mL, EPS-H29 promoted cell proliferation. Significant increase in the HDF cell migration was evident with EPS-H29 (15 μg/mL) treatment in vitro and induced significantly higher (p ≤ 0.0001) closure of the scratch area (90.3 ± 1.1%), compared to the control (84.3 ± 1.3%) at 24 h. Enhanced expression of Ki-67 was associated with the cell proliferative activities of EPS-H29. The animals treated with EPS-H29 showed improved healing of burn wounds with significantly higher wound contraction rate (80.6 ± 9.4%) compared to the positive control (54.6 ± 8.0%) and untreated group (49.2 ± 3.7%) with histopathological evidence of epidermal tissue formation at 15 days of treatment. These results demonstrate the biocompatibility and burn wound healing capability of EPS-H29 and its potential as an effective topical agent for the burn wound care. Graphical Abstract
View
... On the next day, cells were washed with PBS and encountered with 100 μg/mL AO/EtBr solution and immediately observed under FITC (485 excitation /595nm emission) and TRITC (540 excitation /590 nm emission) filter. In merged channel, image cells were observed and up to 300 cells were counted to screen live and dead cells [14,15]. ...
Mitigative Effect of Graphene Oxide Nanoparticle in Maintaining Gut-Liver Homeostasis against Alcohol Injury
Preprint
Full-text available
  • Apr 2024
Alcoholic liver disease (ALD) develops when the immunotolerant environment of the liver is compromised due to excessive alcohol consumption. ALD progression involves variations in the expression of multiple genes, resulting in liver inflammation and the development of a leaky gut. Molecular mechanism involved for ALD progression is still unclear, and due to that there are currently no FDA-approved drugs available for its treatment. In this study, the protective effects of Graphene oxide nanoparticles (GO) was investigated against ethanol-induced damage in the gut-liver axis in in vitro. GO was synthesized using a modified Hummer's method and characterization was done. Given the general concerns regarding nanoparticle toxicity, assessment of cell viability, lipid accumulation, DNA damage, cell death, and the generation of reactive oxygen species (ROS) were conducted using various techniques. Furthermore, gene expression of pro- and anti-inflammatory cytokines was done using RT-qPCR. The findings revealed that GOs promoted cell viability even against ethanol treatment. Additionally, lipid accumulation significantly decreased when cells were treated with GO alongside ethanol compared to ethanol treatment alone, with similar trends observed for other assays. Gene expression analysis indicated that GO treatment reduced the expression of proinflammatory cytokines while enhancing the expression of antioxidant genes. Moreover, GO treatment led to improvements in gut permeability and a reduction in proinflammatory cytokines in colon cells damaged by ethanol. These findings suggest that GO holds promise as a drug carrier, exhibiting no observed toxic effects. By shedding light on the protective effects of GO against ethanol-induced damage, this study contributes to the burgeoning field of nanoparticle-mediated therapy for ALD.
View
... Cell viability and differentiation were assessed in separate cell populations cultured and transfected using the method described above. After a 24h post-transfection incubation period, cell viability was assessed using acridine orange (Sigma-Aldrich, A8097) /ethidium bromide (Sigma-Aldrich , E1510) (AO/EB) staining (Kasibhatla et al. 2006). In brief, the cell media was removed and washed twice with PBS before being stained with 500ul of a PBS solution containing 1:1000 ethidium bromide and 1:1000 acridine orange. ...
Unveiling Ageing-Associated and Caloric Restriction-Associated Changes in miRNA Expression in Rat Skeletal Muscle and the Mechanisms Mediating their Effects on Muscle Cell Function
Preprint
Full-text available
  • Apr 2024
The mechanisms underlying skeletal muscle ageing, whilst poorly understood, are thought to involve dysregulated micro (mi)RNA expression. Using young and aged rat skeletal muscle tissue, we applied high-throughput RNA sequencing to comprehensively study alterations in miRNA expression occurring with age, as well as the impact of caloric restriction (CR) on these changes. Furthermore, the function of the proteins targeted by these age- and CR-associated miRNAs was ascertained. Numerous known and novel age-associated miRNAs were identified of which CR normalised 45.5% to youthful levels. Our results suggested miRNAs upregulated with age to downregulated proteins involved in muscle tissue development and metabolism, as well as longevity pathways, such as AMPK and autophagy. Furthermore, our results found miRNAs downregulated with age to upregulate pro-inflammatory proteins, particularly those involved in innate immunity and the complement and coagulation cascades. Interestingly, CR was particularly effective at normalising miRNAs upregulated with age, rescuing their associated protein coding genes but was less effective at rescuing anti-inflammatory miRNAs downregulated with age. Lastly, the effects of a specific miRNA, miR-96-5p, identified by our analysis to be upregulated with age, were studied in culture C2C12 myoblasts. We demonstrated miR-96-5p to decrease cell viability and markers of mitochondrial biogenesis, myogenic differentiation and autophagy. Overall, our results provide useful information regarding how miRNA expression changes in skeletal muscle, as well as the consequences of these changes and how they are ameliorated by CR.
View
Novel Quaternary Ammonium Aldimine Derivatives Featuring 3,4,5-Trimethoxy Phenyl Fragment: Synthesis, Crystal Structure and Evaluation of Antioxidant and Antibacterial Activity
Article
Full-text available
  • May 2024
This study demonstrates the synthesis of five novel quaternary ammonium aldimines through a two-step synthetic route involving a condensation reaction between 4-pyridincarboxyaldehyde and 3,4,5-trimethoxyaniline, followed by the quaternization of the pyridine N-atom with various aromatic α-bromo ketones. The newly obtained compounds underwent characterization for both purity and molecular structure, utilizing HR-MS, 1D, and 2D NMR spectroscopy in solution, as well as a comparison between single-crystal and powder X-ray analyses in a solid state. The thermal behavior of the studied compounds was evaluated using differential scanning calorimetry (DSC). The antioxidant properties of the compounds were assessed through DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging and ferric-reducing antioxidant power (FRAP) assays, employing Trolox as a standard. The performed in vitro antibacterial screening indicates a selective antibacterial activity against Gram-negative K. pneumoniae and P. aeruginosa, while no such activity is detected for Gram-negative E. coli and Gram-positive S. aureus.
View
View
ResearchGate has not been able to resolve any references for this publication.