caa a22 4500 475775147 CHVBK 20180406123629.0 cr unu---uuuuu 170329e20001101xx s 000 0 eng 10.1007/s00418-004-0712-y doi (NATIONALLICENCE)springer-10.1007/s00418-004-0712-y Calcein-AM is a detector of intracellular oxidative activity [Elektronische Daten] [Jacopo Uggeri, Rita Gatti, Silvana Belletti, Renato Scandroglio, Roberto Corradini, Bianca Rotoli, Guido Orlandini] 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. Springer-Verlag, 2004 Calcein-AM nationallicence ROS nationallicence Confocal microscopy nationallicence Fluorometry nationallicence Uggeri Jacopo Department of Experimental Medicine, Histology Section, University of Parma, Via Volturno 39, 43100, Parma, Italy aut Gatti Rita Department of Experimental Medicine, Histology Section, University of Parma, Via Volturno 39, 43100, Parma, Italy aut Belletti Silvana Department of Experimental Medicine, Histology Section, University of Parma, Via Volturno 39, 43100, Parma, Italy aut Scandroglio Renato Department of Experimental Medicine, Histology Section, University of Parma, Via Volturno 39, 43100, Parma, Italy aut Corradini Roberto Department of Organic and Industrial Chemistry, University of Parma, Parco Area delle Scienze 17/A, 43100, Parma, Italy aut Rotoli Bianca Department of Experimental Medicine, General and Clinical Pathology Section, University of Parma, Via Volturno 39, 43100, Parma, Italy aut Orlandini Guido Department of Experimental Medicine, Histology Section, University of Parma, Via Volturno 39, 43100, Parma, Italy aut Histochemistry and Cell Biology Springer-Verlag 122/5(2000-11-01), 499-505 0948-6143 122:5<499 2000 122 418 https://doi.org/10.1007/s00418-004-0712-y text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s00418-004-0712-y text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Uggeri Jacopo Department of Experimental Medicine, Histology Section, University of Parma, Via Volturno 39, 43100, Parma, Italy aut NATIONALLICENCE 700 1- Gatti Rita Department of Experimental Medicine, Histology Section, University of Parma, Via Volturno 39, 43100, Parma, Italy aut NATIONALLICENCE 700 1- Belletti Silvana Department of Experimental Medicine, Histology Section, University of Parma, Via Volturno 39, 43100, Parma, Italy aut NATIONALLICENCE 700 1- Scandroglio Renato Department of Experimental Medicine, Histology Section, University of Parma, Via Volturno 39, 43100, Parma, Italy aut NATIONALLICENCE 700 1- Corradini Roberto Department of Organic and Industrial Chemistry, University of Parma, Parco Area delle Scienze 17/A, 43100, Parma, Italy aut NATIONALLICENCE 700 1- Rotoli Bianca Department of Experimental Medicine, General and Clinical Pathology Section, University of Parma, Via Volturno 39, 43100, Parma, Italy aut NATIONALLICENCE 700 1- Orlandini Guido Department of Experimental Medicine, Histology Section, University of Parma, Via Volturno 39, 43100, Parma, Italy aut NATIONALLICENCE 773 0- Histochemistry and Cell Biology Springer-Verlag 122/5(2000-11-01), 499-505 0948-6143 122:5<499 2000 122 418 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer