Experimental system for quantifying DSBs in single, living cells. (A) Schematic drawing of the 53BP1 reporter. (B-C) Cells expressing 53BP1-mCherry were fixed and stained with anti γ-H2AX antibody 30 minutes and 2 hours after 5Gy γ-irradiation. The overlaid image, and the measured intensities of both 53BP1-mCherry and γ-H2AX staining across a line in the nucleus (C) show co-localization between 53BP1 and γ-H2AX foci. (D) Time-lapse images of a cell expressing 53BP1-mCherry after 5Gy γ-irradiation. Images are maximum projections of z-stacks through the nucleus (see Methods section) in the mCherry channel. (E) Example of the automated segmentation for the enumeration of 53BP1-mCherry foci in a cell. Segmented foci are indicated as red circles. Image processing was performed using custom written Matlab based software (see Methods section for algorithmic details). (F) Enumerated 53BP1-mCherry foci for three cells using five different thresholds for foci detection. Except for very low levels, the quantification of foci is robust to changes in the threshold. (G) Enumerated 53BP1-mCherry foci (dots) and exponential fits to the raw data (dashed lines) for two cells. (H-I) Distribution of the initial number (H) and half-life (I) of 53BP1 foci in a population of cells treated with 5Gy γ-irradiation. The analysis was performed using a range of 0.6 to 1.3 times the optimal threshold level. Error bars indicate the standard deviation of the analyses performed at different threshold levels. Number of cells = 97. (J-K) Cell cycle distributions of untreated cells (0Gy) and irradiated cells (10Gy) showing a strong cell cycle arrest post irradiation with minimal death (sub G1 fraction). DSBs double strand breaks.
Loewer et al. BMC Biology 2013 11:114 doi:10.1186/1741-7007-11-114