Nature Methods
- 5, 261 - 266 (2008)
Published online: 28 February 2008; | doi:10.1038/nmeth.f.206
Induction of linear tracks of DNA double-strand breaks by  -particle irradiation of cellsJan Stap1, 4, Przemek M Krawczyk1, 4, Carel H Van Oven1, Gerrit W Barendsen2, Jeroen Essers3, Roland Kanaar3 & Jacob A Aten11
Center for Microscopical Research, Department of Cell Biology and Histology, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. 2
Department of Radiotherapy, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. 3
Department of Cell Biology and Genetics, Cancer Genomics Center and Department of Radiation Oncology, Erasmus Medical Center, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands. 4
These authors contributed equally to this work.
Correspondence should be addressed to Jan Stap j.stap@amc.uva.nl Understanding how cells maintain genome integrity when challenged with DNA double-strand breaks (DSBs) is of major importance, particularly since the discovery of multiple links of DSBs with genome instability and cancer-predisposition disorders1,
2. Ionizing radiation is the agent of choice to produce DSBs in cells3; however, targeting DSBs and monitoring changes in their position over time can be difficult. Here we describe a procedure for induction of easily recognizable linear arrays of DSBs in nuclei of adherent eukaryotic cells by exposing the cells to  particles from a small Americium source (Box 1). Each particle traversing the cell nucleus induces a linear array of DSBs, typically 10–20 DSBs per 10  m track length4. Because particles cannot penetrate cell-culture plastic or coverslips, it is necessary to irradiate cells through a Mylar membrane. We describe setup and irradiation procedures for two types of experiments: immunodetection of DSB response proteins in fixed cells grown in Mylar-bottom culture dishes (Option A) and detection of fluorescently labeled DSB-response proteins in living cells irradiated through a Mylar membrane placed on top of the cells (Option B). Using immunodetection, recruitment of repair proteins to individual DSB sites as early as 30 s after irradiation can be detected. Furthermore, combined with fluorescence live-cell microscopy of fluorescently tagged DSB-response proteins, this technique allows spatiotemporal analysis of the DSB repair response in living cells. Although the procedures might seem a bit intimidating, in our experience, once the source and the setup are ready, it is easy to obtain results. Because the live-cell procedure requires more hands-on experience, we recommend starting with the fixed-cell application.
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