X-irradiation of cells on glass slides has a dose doubling impact

Immunofluorescence detection of gamma H2AX foci is a widely used tool to quantify the induction and repair of DNA double-strand breaks (DSBs) induced by ionising radiation. We observed that X-irradiation of mammalian cells exposed on glass slides induced twofold higher foci numbers compared to irradiation with gamma-rays. Here, we show that the excess gamma H2AX foci after X-irradiation are produced from secondary radiation particles generated from the irradiation of glass slides. Both 120kV X-rays and Cs-137 gamma-rays induce similar to 20 gamma H2AX foci per Gy in cells growing on thin (similar to 2 mu m) plastic foils immersed in water. The same yield is obtained following gamma-irradiation of cells growing on glass slides. However, 120kV X-rays produce similar to 40 gamma H2AX foci per Gy in cells growing on glass, twofold greater than obtained using cells irradiated on plastic surfaces. The same increase in gamma H2AX foci number is obtained if the plastic foil on which the cells are grown is irradiated on a glass slide. Thus, the physical proximity to the glass material and not morphological differences of cells growing on different surfaces accounts for the excess gamma H2AX foci. The increase in foci number depends on the energy and is considerably smaller for 25 W relative to 120 kV X-rays, a finding which can be explained by known physical properties of radiation. The kinetics for the loss of foci, which is taken to represent the rate of DSB repair, as well as the Artemis dependent repair fraction, was similar following X- or gamma-irradiation, demonstrating that DSBs induced by this range of treatments are repaired in an identical manner. (c) 2007 Elsevier B.V. All rights reserved.