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Background <p>Acridine orange (AO) is a membrane-permeable fluorescent dye capable of staining and distinguishing DNA, nuclear RNA, and various cytoplasmic organelles in cells. AO stains DNA and nuclear RNA green, while acidic organelles such as lysosomes and endosomes appear red. These staining characteristics make AO a valuable tool for assessing cell and tissue viability, lysosomal membrane integrity, and detecting tumor cells. However, its application in aquatic invertebrates remains limited, and no standardized protocol currently exists for small crustaceans.</p> Objective <p>In this protocol manuscript, we present an optimized AO staining protocol for a mysid model, including recommendations for effective AO concentrations, based on responses to both organic and inorganic toxicants, such as tralopyril and cadmium.</p> Result <p>Following exposure to the two toxicants, cellular toxicity responses—such as lysosomal membrane destabilization and cell death—were successfully evaluated by measuring green and red fluorescence intensities in the mysid model.</p> Conclusion <p>The optimized AO staining technique in mysids allows for a rapid, visual, and quantitative evaluation of cellular damage. This method, along with initial procedural guidelines, has the potential to be adapted for application in other small crustacean species.</p>

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An optimized method for estimating toxicity and cell death in the mysid model using acridine orange staining

  • 期刊论文

  • 发表日期：

    2025年7月24日

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  • Somyeong Lee,
  • Jae-Sung Rhee

摘要

Background

Acridine orange (AO) is a membrane-permeable fluorescent dye capable of staining and distinguishing DNA, nuclear RNA, and various cytoplasmic organelles in cells. AO stains DNA and nuclear RNA green, while acidic organelles such as lysosomes and endosomes appear red. These staining characteristics make AO a valuable tool for assessing cell and tissue viability, lysosomal membrane integrity, and detecting tumor cells. However, its application in aquatic invertebrates remains limited, and no standardized protocol currently exists for small crustaceans.

Objective

In this protocol manuscript, we present an optimized AO staining protocol for a mysid model, including recommendations for effective AO concentrations, based on responses to both organic and inorganic toxicants, such as tralopyril and cadmium.

Result

Following exposure to the two toxicants, cellular toxicity responses—such as lysosomal membrane destabilization and cell death—were successfully evaluated by measuring green and red fluorescence intensities in the mysid model.

Conclusion

The optimized AO staining technique in mysids allows for a rapid, visual, and quantitative evaluation of cellular damage. This method, along with initial procedural guidelines, has the potential to be adapted for application in other small crustacean species.