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    JC-10 probe as a novel method for analyzing the mitochondrial membrane potential and cell stress in whole zebrafish embryos

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    JC-10 probe as a novel method for analyzing the mitochondrial membrane potential and cell stress in whole zebrafish embryos.pdf (859.5Kb)
    Date
    2021-12
    Author
    Younes, Nadin
    Alsahan, Bana S.
    Al-Mesaifr, Asmaa J.
    Da'as, Sahar I.
    Pintus, Gianfranco
    Majdalawieh, Amin F.
    Nasrallah, Gheyath K.
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    Abstract
    Background: A sensitive method to investigate cellular stress and cytotoxicity is based on measuring mitochondrial membrane potential. Recently, JC-10, was developed to measure mitochondrial membrane potential in vitro and used as an indicator for cytotoxicity. Yet, JC-10 has never been used in vivo (whole organism). In normal cells, JC-10 concentrates in the mitochondrial matrix, where it forms red fluorescent aggregates. However, in apoptotic/necrotic cells, JC-10 diffuses out of the mitochondria, changes to monomeric form, and stains cells in green. Here, we aimed to develop and optimize a JC-10 assay to measure cytotoxicity in zebrafish embryo. We also investigated the effectiveness of JC-10 assay by comparing it to common cytotoxicity assays. Methods: Zebrafish embryos were exposed to a toxic surfactant AEO-7 at no observed effect concentration (6.4 μg/L), and then cytotoxicity was measured using (i) JC-10 mitochondrial assay, (ii) acridine orange (AO), (iii) TUNEL assay, and (iv) measuring the level of Hsp70 by western blotting. Results: As compared to the negative control, embryos treated with NOEC of AEO-7 did not show significant cytotoxicity when assessed by AO, TUNEL or western blotting. However, when JC-10 was used under the same experimental conditions, a significant increase of green:red fluorescent ratio signal was detected in the AEO-7 treated embryos, indicating mitochondrial damage and cellular cytotoxicity. Noteworthy, the observed green: red ratio increase was dose dependent, suggesting specificity of the JC-10 assay. Conclusion: JC-10 is a sensitive in vivo method, thus, can be used as surrogate assay to measure cytotoxicity in whole zebrafish embryos.
    DOI/handle
    http://dx.doi.org/10.1093/toxres/tfab114
    http://hdl.handle.net/10576/25638
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    • Biomedical Sciences [‎847‎ items ]

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