Protocol abstract
Nature Protocols 2, - 3045 - 3057 (2007)
Published online: 21 November 2007 | doi:10.1038/nprot.2007.420
Subject Categories: Cell and developmental biology | Cell and tissue culture | Model organisms | Pharmacology and toxicology | Spectroscopy and structural analysis
Measurement of cell proliferation by heavy water labeling
Robert Busch1,4,6, Richard A Neese2,6, Mohamad Awada1,5, Gregory M Hayes1 & Marc K Hellerstein2,3
Abstract
DNA replication occurs almost exclusively during S-phase of the cell cycle and represents a simple biochemical metric of cell division. Previous methods for measuring cell proliferation rates have important limitations. Here, we describe experimental protocols for measuring cell proliferation and death rates based on the incorporation of deuterium (2H) from heavy water (2H2O) into the deoxyribose moiety of purine deoxyribonucleotides in DNA of dividing cells. Label incorporation is measured by gas chromatography/mass spectrometry. Modifications of the basic protocol permit analysis of small cell samples (down to 2,000 cells). The theoretical basis and operational requirements for effective use of these methods to measure proliferation and death rates of cells in vivo are described. These methods are safe for use in humans, have technical and interpretation advantages over alternative techniques and can be used on small numbers of cells. The protocols enable definitive in vivo studies of the fraction or absolute number of newly divided cells and their subsequent survival kinetics in animals and humans.
- KineMed Inc., 5980 Horton Street, Emeryville, California 94608, USA.
- Department of Nutritional Sciences and Toxicology, University of California at Berkeley, Berkeley, California 94720, USA.
- Department of Medicine, University of California at San Francisco, San Francisco, California 94110, USA.
- Present address: Department of Medicine, University of Cambridge, Cambridge, CB2 2QQ, UK.
- Present address: ThermoFisher Scientific, HyClone Laboratories, Logan, Utah 84321, USA.
- These authors contributed equally to this work.
Correspondence to: Marc K Hellerstein2,3 e-mail: march@nature.berkeley.edu
nature-products
A-Z product listing
- DB-17 column(Agilent)
- DNA extraction kits(Qiagen)
- dR(Sigma)
- GC/MS instrument(Agilent)
- Pentafluorobenzyl hydroxylamine(Sigma-Aldrich)
- Potato acid phosphatase(Calbiochem)
