Characterization of chromatin domains by 3D fluorescence microscopy: An automated methodology for quantitative analysis and nuclei screening

Authors

  • Sylvain Cantaloube,

    1. Institut Curie, Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche UMR218, Laboratory of Nuclear Dynamics and Genome Plasticity, Paris Cedex, France
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  • Kelly Romeo,

    1. Institut Curie, Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche UMR218, Laboratory of Nuclear Dynamics and Genome Plasticity, Paris Cedex, France
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  • Patricia Le Baccon,

    1. Institut Curie, Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche UMR218, Laboratory of Nuclear Dynamics and Genome Plasticity, Paris Cedex, France
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  • Geneviève Almouzni,

    1. Institut Curie, Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche UMR218, Laboratory of Nuclear Dynamics and Genome Plasticity, Paris Cedex, France
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  • Jean-Pierre Quivy

    Corresponding author
    1. Institut Curie, Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche UMR218, Laboratory of Nuclear Dynamics and Genome Plasticity, Paris Cedex, France
    • Institut Curie, Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche UMR218, Laboratory of Nuclear Dynamics and Genome Plasticity, Paris Cedex, France.
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Abstract

Fluorescence microscopy has provided a route to qualitatively analyze features of nuclear structures and chromatin domains with increasing resolution. However, it is becoming increasingly important to develop tools for quantitative analysis. Here, we present an automated method to quantitatively determine the enrichment of several endogenous factors, immunostained in pericentric heterochromatin domains in mouse cells. We show that this method permits an unbiased characterization of changes in the enrichment of several factors with statistical significance from a large number of nuclei. Furthermore, the nuclei can be sorted according to the enrichment value of these factors. This method should prove useful to monitor events related to changes in the amount, rather than the presence or absence, of any factor. By adapting a few parameters, it could be extended to other nuclear structures and the benefit of using available software will permit its use in many biological labs.

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