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| Content Provider | World Health Organization (WHO)-Global Index Medicus |
|---|---|
| Author | Stap, Jan Krawczyk, Przemek M. Van Oven, Carel H. Barendsen, Gerrit W. Essers, Jeroen Kanaar, Roland Aten, Jacob A. |
| Description | Country affiliation: Netherlands Author Affiliation: Stap J ( Center for Microscopical Research, Department of Cell Biology and Histology, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. j.stap@amc.uva.nl) |
| Abstract | 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 disorders. Ionizing radiation is the agent of choice to produce DSBs in cells; 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 alpha particles from a small Americium source (Box 1). Each alpha particle traversing the cell nucleus induces a linear array of DSBs, typically 10-20 DSBs per 10 mum track length. Because alpha 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. |
| File Format | HTM / HTML |
| ISSN | 15487091 |
| Issue Number | 3 |
| Volume Number | 5 |
| e-ISSN | 15487105 |
| Journal | Nature Methods |
| Language | English |
| Publisher | Nature Publishing Group |
| Publisher Date | 2008-03-01 |
| Publisher Place | United States |
| Access Restriction | One Nation One Subscription (ONOS) |
| Subject Keyword | Discipline Clinical Laboratory Techniques Alpha Particles Dna Damage Dna Radiation Effects Americium Cell Line, Tumor Humans Journal Article Research Support, Non-u.s. Gov't |
| Content Type | Text |
| Resource Type | Article |
| Subject | Cell Biology Biochemistry Molecular Biology Biotechnology |
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