Calcein-AM is a detector of intracellular oxidative activity.
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.
Uggeri
Jacopo
J
Department of Experimental Medicine, Histology Section, University of Parma, Via Volturno 39, 43100 Parma, Italy. jacopo.uggeri@unipr.it
Gatti
Rita
R
Belletti
Silvana
S
Scandroglio
Renato
R
Corradini
Roberto
R
Rotoli
Bianca M
BM
Orlandini
Guido
G
D002474:Cell-Free System; D002478:Cells, Cultured; D002855:Chromatography, Thin Layer; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D005470:Fluorometry; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D018613:Microscopy, Confocal; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D019820:Xanthine / Q000494:pharmacology; D014969:Xanthine Oxidase / Q000494:pharmacology
D005452:Fluoresceins; D005456:Fluorescent Dyes; D016877:Oxidants; D017382:Reactive Oxygen Species; C085925:calcein AM; D019820:Xanthine; D006861:Hydrogen Peroxide; D014969:Xanthine Oxidase; D013755:Tetradecanoylphorbol Acetate