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Detection of S-phase cells in tissue sections by in situ DNA replication

Nature Cell Biology volume 2, pages 244–245 (2000)Cite this article

Here we demonstrate a rapid and simple method for identifying S-phase cells in tissue sections or cell cultures without the need to prelabel them with radioactive or halogenated precursors before sample collection. When sections of snap-frozen tissue samples or frozen, permeabilized cell cultures grown on coverslips are incubated in a buffer containing ribonucleoside triphosphates and deoxyribonucleoside triphosphates, S-phase cells can be identified because their nuclei continue to synthesize DNA in situ. Incubations as short as 15 min allow the detection of S-phase cells by fluorescent microscopy. This method has many potential applications in cell and developmental biology and in medicine, especially cancer diagnosis.

While developing cell-free DNA-replication systems from human cells, we observed that frozen nuclei from S-phase cells continue to synthesize DNA in vitro after they are rethawed and incubated in buffers containing precursors1,2,3. The method described here exploits this observation to detect S-phase cells in frozen tissue sections by virtue of their DNA replication in situ.

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Figure 1: DNA replication in situ in permeabilized HeLa cells — nuclei and intranuclear sites labelled in vitro are the same as those that were labelled in vivo.
Figure 2: The identification of S-phase cells in frozen tissue sections by virtue of their DNA replication in situ is potentially useful in diagnosing cancer.

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Acknowledgements

We thank K. Bird and P. Laskey for technical assistance, A. Freeman for histological sections, and D. Coverley, G. de la Cueva Mendez, D. Hatton, T. Krude and D. Santamaria Velilla for discussions on the manuscript. All human tissue used was obtained at Addenbrooke’s Hospital, Cambridge, with Local Research Ethics Committee approval. This work was supported by the Cancer Research Campaign and the Louis Jeantet Foundation.

Correspondence and requests for materials should be addressed to A.D.M.

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Authors and Affiliations

  1. Wellcome/CRC Institute, Tennis Court Road,, Cambridge, CB2 1QR, UK

    Anthony D. Mills & Ronald A. Laskey

  2. Department of Zoology, University of Cambridge, Downing St, CB2 3EJ, Cambridge, UK

    Anthony D. Mills & Ronald A. Laskey

  3. Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP, UK

    Nicholas Coleman & Lesley S. Morris

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  1. Anthony D. Mills
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Correspondence to Anthony D. Mills.

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Mills, A., Coleman, N., Morris, L. et al. Detection of S-phase cells in tissue sections by in situ DNA replication. Nat Cell Biol 2, 244–245 (2000). https://doi.org/10.1038/35008670

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