Abstract
Flow cytometry is an essential tool to monitor DNA content and determine cell cycle distribution. Its utility in fission yeast reflects the ease of sample preparation, the stochiometric binding of the most popular DNA dyes (propidium iodide and Sytox Green), and ability to monitor cell size. However, the study of DNA replication with multicolour flow analysis has lagged behind its use in mammalian cells. We present basic and advanced protocols for analysis of DNA replication in fission yeast by flow cytometry including whole cell, nuclear “ghosts,” two-color imaging with BrdU, and estimates of DNA synthesis using EdU.
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Acknowledgments
Thanks to Scott Clarke at Molecular Probes, Invitrogen (Life Technologies) for advice on modifying EdU protocols; David Chambers of the Center for Cell and Molecular Imaging at the Salk Institute for helpful advice; instructors at the Annual Research Course in Cytometry (2011); and Oscar Aparicio at USC for sharing his FACScan. Supported by grant NIGMS R01 081418 to S.L.F.
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Sabatinos, S.A., Forsburg, S.L. (2015). Measuring DNA Content by Flow Cytometry in Fission Yeast. In: Vengrova, S., Dalgaard, J. (eds) DNA Replication. Methods in Molecular Biology, vol 1300. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2596-4_5
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DOI: https://doi.org/10.1007/978-1-4939-2596-4_5
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2595-7
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