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  Institute of Cell and Molecular Science
Flow Cytometry Core Facility equipment, images used with permission by BD
  Flow Cytometry Core Facility
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Cell cycle analysis


Flow cytometry may be used to analyse cellular proliferation by determining the stages of the cell cycle within a population of cells. This can be carried out on fixed or live cells using different fluorescent DNA binding dyes in conjunction with monoclonal antibodies to analyse antigen expression or with FP expressing cells.

The rate of cell division can be monitored by pulsing cells with BrdU and harvesting at different times. After labelling with anti-BrdU-FITC, RNAse treatment and PI staining, cells synthesize DNA during the BrdU pulse will positive for FITC.

The use of EdU instead of BrdU to label newly synthesized DNA via click copper chemistry covalently binding fluorescent azide allows the investigator to label surface and or intracellular antigens more readily and rapidly than via the use of coarse treatment required to label BrdU.

Histone H3 is phosphorylated from prophase to anaphase during mammalian cell mitosis and meiosis. H3 can thus be used to determine the percentage of cells in m phase and thus can differentiate between G2 and m phases when performing cell cycle analysis by flow cytometry.

Senescent or Go cells may be identified by their low RNA content compared to cells in G1 by the use of Pyronin Y which fluoresces at 575 nm when bound to RNA. Thus when used in conjunction with Hoechst 33342 Go and G1 cells can be discriminated.

The time at which genes are replicated during S phase can be investigated by first pulsing cells with BrdU, fixing in 70% ethanol and staining with PI after RNAse treatment. On sorting, S phase is divided into 4 sort fractions by gating S phase into early, intermediate and late S areas. DNA with BrdU is then immunoprecipitated and PCR carried out to the time at which specific genes of interest have replicated.


P Takousis, P Johonnett, J Williamson, P Sasieni, G Warnes, T Forshew, V Azuara, A Fisher, PJ Wu, PJ Jones, R Vatcheva, S Beck, D Sheer. Replication timing profile reflects the distinct functional and genomic features of the MHC Class II Region. Cell Cycle Oct 6:19, 2393-2398, 2007.

V Azuara, P Perry, S Sauer, M Spivakov, HF Jorgensen, RM John, M Gouti, M Casanova, G Warnes, M Merkenschlager, AG Fisher. Chromatin signatures of pluripotent cell lines. Nature Cell Biol. 8, 532-538, 2006.

P Perry, S Saur, N Billion, WD Richardson, M Spivakov, G Warnes, FJ Livesey, M Merkenschlager, AG Fisher, V Auara. A dynamic switch in the replication timing of key regulator genes in embryonic stem cells upon neural induction. Cell Cycle 3, (12), 1645-50, 2004.

by Gary Warnes. © Queen Mary, University of London 2007
Institute of Cell and Molecular Science, The Blizard Building, Barts and The London School of Medicine and Dentistry, 4 Newark Street, London E1 2AT, UK Tel: +44 (0)20 7882 2483, Fax: +44 (0)20 7882 2200