The reported method allows for a simple and rapid monitoring of DNA replication and cell cycle progression in eukaryotic cells in vitro. The DNA of replicating cells is labeled by incorporation of a metabolically-active fluorescent (Cy3) deoxyuridine triphosphate derivative, which is delivered into the cells by a synthetic transporter (SNTT1). The cells are then fixed, stained with DAPI and analyzed by flow cytometry. Thus, this protocol obviates post-labeling steps, which are indispensable in currently used incorporation assays (BrdU, EdU). The applicability of the protocol is demonstrated in analyses of cell cycles of adherent (U-2 OS, HeLa S3, RAW 264.7, J774 A.1, Chem-1, U-87 MG) and suspension (CCRF-CEM, MOLT-4, THP-1, HL-60, JURKAT) cell cultures, including those affected by a DNA polymerase inhibitor (aphidicolin). Owing to a short incorporation time (5-60 min) and reduced number of steps, the protocol can be completed within 1-2 h with a minimal cell loss and with excellent reproducibility.

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Flemingovo Nam 2 CZ 16610 Prague 6 Czech Republic

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Flemingovo Nam 2 CZ 16610 Prague 6 Czech Republic; University of Cambridge Department of Chemistry Lensfield Road Cambridge CB2 1EW UK

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trans-Cyclooctene- and Bicyclononyne-Linked Nucleotides for Click Modification of DNA with Fluorogenic Tetrazines and Live Cell Metabolic Labeling and Imaging