Document Detail


Induction of linear tracks of DNA double-strand breaks by alpha-particle irradiation of cells.
MedLine Citation:
PMID:  18309310     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Jan Stap; Przemek M Krawczyk; Carel H Van Oven; Gerrit W Barendsen; Jeroen Essers; Roland Kanaar; Jacob A Aten
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Nature methods     Volume:  5     ISSN:  1548-7105     ISO Abbreviation:  Nat. Methods     Publication Date:  2008 Mar 
Date Detail:
Created Date:  2008-02-29     Completed Date:  2008-03-13     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101215604     Medline TA:  Nat Methods     Country:  United States    
Other Details:
Languages:  eng     Pagination:  261-6     Citation Subset:  IM    
Affiliation:
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
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MeSH Terms
Descriptor/Qualifier:
Alpha Particles*
Americium
Cell Line, Tumor
DNA / radiation effects*
DNA Damage*
Humans
Chemical
Reg. No./Substance:
7440-35-9/Americium; 9007-49-2/DNA

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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