Linked Life Data

18309310

Source:http://linkedlifedata.com/resource/pubmed/id/18309310

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
3
pubmed:dateCreated
2008-2-29
pubmed:abstractText
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.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Mar
pubmed:issn
1548-7105
pubmed:author
pubmed:issnType
Electronic
pubmed:volume
5
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
261-6
pubmed:meshHeading
pubmed:year
2008
pubmed:articleTitle
Induction of linear tracks of DNA double-strand breaks by alpha-particle irradiation of cells.
pubmed: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
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't