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Calcein-AM is a detector of intracellular oxidative activity

Histochemistry and Cell Biology volume 122, pages 499–505 (2000)Cite this article

Abstract

Calcein-acetoxymethylester (calcein-AM) is a non-fluorescent, cell permeant compound, which is converted by intracellular esterases into calcein, an anionic fluorescent form. It is used in microscopy and fluorometry and provides both morphological and functional information of viable cells. In this study we have tested the response of calcein-AM to oxidation. In cell-free fluorometric assays, H2O2 and xanthine–xanthine oxidase induced a dose-dependent emission of the AM form but had no effects on calcein. Fluorometric and confocal microscopy tests on human fibroblasts confirmed that the cell permeant AM form is the actual sensor since its removal from culture medium, and its consequent back-diffusion, made the system insensitive to oxidative stimuli. In time-lapse confocal microscopy, calcein-AM detected changes in the intracellular redox state following direct oxidation (H2O2, xanthine–xanthine oxidase) and phorbol ester treatment. Comparative tests showed that calcein-AM sensitivity to oxidation is about one order of magnitude higher than other fluorescein derivatives. The absence of leakage, due to the presence of the probe in the extracellular compartment, and its low toxicity allow to perform experiments for prolonged times following the response to the same or different stimuli repeatedly applied. We propose calcein-AM as a sensitive tool for intracellular ROS generation in living cells with useful applications for real-time imaging in confocal microscopy.

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Acknowledgements

The confocal microscope is a facility of CIM, Centro Interfacoltà Misure, University of Parma, Italy. This study was supported by a local research grant (FIL, University of Parma, Italy).

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Affiliations

  1. Department of Experimental Medicine, Histology Section, University of Parma, Via Volturno 39, 43100, Parma, Italy

    Jacopo Uggeri, Rita Gatti, Silvana Belletti, Renato Scandroglio & Guido Orlandini

  2. Department of Organic and Industrial Chemistry, University of Parma, Parco Area delle Scienze 17/A, 43100, Parma, Italy

    Roberto Corradini

  3. Department of Experimental Medicine, General and Clinical Pathology Section, University of Parma, Via Volturno 39, 43100, Parma, Italy

    Bianca Maria Rotoli

Authors
  1. Jacopo Uggeri
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  2. Rita Gatti
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  3. Silvana Belletti
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  4. Renato Scandroglio
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  5. Roberto Corradini
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  6. Bianca Maria Rotoli
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  7. Guido Orlandini
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Corresponding author

Correspondence to Jacopo Uggeri.

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Uggeri, J., Gatti, R., Belletti, S. et al. Calcein-AM is a detector of intracellular oxidative activity. Histochem Cell Biol 122, 499–505 (2000). https://doi.org/10.1007/s00418-004-0712-y

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  • DOI: https://doi.org/10.1007/s00418-004-0712-y

Keywords

  • Calcein-AM
  • ROS
  • Confocal microscopy
  • Fluorometry