We are analyzing https://link.springer.com/article/10.1007/s00418-004-0712-y.

Title:
Calcein-AM is a detector of intracellular oxidative activity | Histochemistry and Cell Biology
Description:
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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p-glycoprotein-related multidrug resistance month download article/chapter gap-junctional coupling measured time-lapse confocal microscopy real-time imaging intracellular calcium analysis related subjects xanthine–xanthine oxidase induced cell-free fluorometric assays intracellular oxidative activity intracellular ros generation full article pdf intracellular redox state privacy choices/manage cookies confocal imaging local research grant scanning laser microscopy xanthine–xanthine oxidase confocal laser microscopy confocal microscopy tests brenner da mitochondrial permeability transition fluorescence 10 cell biology aims human fibroblasts confirmed human colon cells european economic area apoptotic phenomena induced reactive oxygen species reactive species formation cell-cell communication nitric oxide generation necrotic cell killing oxidative product formation induced oxidative stress dose-dependent emission consequent back-diffusion comparative tests showed automatic hotspots detection early vital markers receptor-ligand internalization centro interfacoltà misure article histochemistry calcium mobilizations cell permeant compound free radical damage conditions privacy policy detect oxidative stress stimuli repeatedly applied sensitive transcription factors

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         description: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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         Biochemistry
         Developmental Biology
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