TY - JOUR
T1 - Induction of linear tracks of DNA double-strand breaks by alpha-particle irradiation of cells
AU - Stap, Jan
AU - Krawczyk, Przemek M.
AU - van Oven, Carel H.
AU - Barendsen, Gerrit W.
AU - Essers, Jeroen
AU - Kanaar, Roland
AU - Aten, Jacob A.
PY - 2008
Y1 - 2008
N2 - 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(1,2). Ionizing radiation is the agent of choice to produce DSBs in cells(3); 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 a particles from a small Americium source (Box 1). Each a particle traversing the cell nucleus induces a linear array of DSBs, typically 10-20 DSBs per 10 mu m track length(4). Because a 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
AB - 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(1,2). Ionizing radiation is the agent of choice to produce DSBs in cells(3); 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 a particles from a small Americium source (Box 1). Each a particle traversing the cell nucleus induces a linear array of DSBs, typically 10-20 DSBs per 10 mu m track length(4). Because a 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
U2 - https://doi.org/10.1038/nmeth.f.206
DO - https://doi.org/10.1038/nmeth.f.206
M3 - Article
C2 - 18309310
SN - 1548-7091
VL - 5
SP - 261
EP - 266
JO - Nature methods
JF - Nature methods
IS - 3
ER -