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Continuing progress in super-resolution microscopy enables the study of sub-chromosomal chromatin organization in single cells with unprecedented detail. Here we describe refined methods for pulse-chase replication labeling of individual chromosome territories (CTs) and replication domain units in mammalian cell nuclei, with specific focus on their application to three-dimensional structured illumination microscopy (3D-SIM). We provide detailed protocols for highly efficient electroporation-based delivery or scratch loading of cell-impermeable fluorescent nucleotides for live-cell studies. Furthermore, we describe the application of (2'S)-2'-deoxy-2'-fluoro-5-ethynyluridine (F-ara-EdU) and 5-vinyl-2'-deoxyuridine (VdU) for the in situ detection of segregated chromosome territories and sister chromatids with minimized cytotoxic side effects.

Original publication

DOI

10.1007/978-1-0716-2221-6_9

Type

Journal article

Journal

Methods Mol Biol

Publication Date

2022

Volume

2476

Pages

111 - 128

Keywords

Chromatin, Chromosome territories, Replication domains, Replication labeling, Structured illumination microscopy, Super-resolution imaging, Animals, Cell Nucleus, Chromatids, Chromatin, Mammals, Microscopy