Quantification of DNA Double-Strand Breaks

Directly after irradiation, 1 × 10⁴ cells per well (1.8 cm²) were seeded in duplicate in chamber slides (LabTek®, Nunc, Roskilde, Denmark) and incubated for 24 h. After fixation with 2% formaldehyde and permeabilisation with 0.25% triton-X 100 (both Sigma Aldrich Chemie GmbH, Munich, Germany) the cells were consecutively incubated 60 min with anti-γH2AX antibody (1:500, clone JBW301, Merck Millipore) and Alexa Fluor 594 goat anti-mouse IgG1 (1:400, Molecular Probes®/Life Technologies, Darmstadt, Germany) for 30 min. The slides were mounted with Vectashield® containing anti-4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Inc., Burlingame, CA). The foci were visualised with an Eclipse TE300 inverted microscope (Nikon, Tokyo, Japan). At a magnification of 1000×, the foci of 50 cells per chamber were counted; two chambers per irradiation. The extra yield (∆Y) was calculated as the difference between irradiated samples and the individual 0 Gy control value of residual foci as a function of dose and plotted in a graph. Linearisation was performed as described by Barendsen [30 (link)]. RBE values, referred to as RBE_{(foci 24 h)} in the text, were calculated with respect to LDR X-rays via the α value with reference to Franken et al. [31 (link), 32 (link)]. Fits to the data points using the eq. F(D) = αD + βD² yielded β values of zero.