Therefore, a range of both open access and commercial programs were developed for automatic foci detection. This is often criticized for being time-consuming and operator-biased. The conventional method of foci counting in microscopy images is manual counting. However, this also requires sufficient image magnification and accurate image processing. The main advantage of this approach is the ability to provide quantitative information about the number of foci in single cells and, thus, the number of DSBs. One of them is immunofluorescent labelling of γH2AX and nuclei followed by microscopy imaging and analysis. Automatic methods for γH2AX foci quantificationĪ wide variety of experimental techniques has been developed for the detection of γH2AX foci in cultured cells. Therefore, the quantification of γH2AX foci is widely used for estimating the number of DSBs and applied for modelling and understanding DNA damage repair processes in cells. For mammalian cells the number of foci per cell increases with respect to DNA damage level roughly by a rate of 20–40 DSB foci per nucleus per Gy measured 30 min after irradiation. Recently, a direct correlation between the number of γH2AX foci and the number of DSBs was established. γH2AX foci can be visualized in single cells using fluorescence microscopy (see below). Phosphorylated H2AX (γH2AX) accumulate at DSBs sites creating a focus, which is required for the assembly of DNA damage repair proteins. However, a key event prompting the DNA damage response is the phosphorylation of serine 139 of H2AX molecules, a histone H2A variant, on chromatin flanking DSB sites. įor repairing DSBs cells utilize several repair pathways. DNA double-strand breaks (DSBs) are the most severe type of DNA damage, because their inaccurate repair can cause chromosomal translocations possibly leading to genomic instability and cancer development.
ΓH2AX foci as DNA damage response measureĪ variety of genotoxic stresses including ionizing radiation (IR) induce DNA damage. ConclusionsįoCo is an open-source user-friendly software with GUI for individual foci counting, which is able to produce reliable and robust foci quantifications even for low signal/noise ratios and densely distributed foci. FoCo requires only 5 parameters that have to be adjusted by the user. FoCo has the following advantages with respect to other software packages: i) the ability to reliably quantify even densely distributed foci, e.g., on images of cells subjected to radiation doses up to 10 Gy, ii) robustness of foci quantification in the sense of suppressing out-of-focus background signal, and iii) its simplicity. In this study, we present the algorithm FoCo for reliable and robust automatic nuclear foci counting in single cell images. One of the experimental techniques for γH2AX detection in cultured cells is immunofluorescent labelling of γH2AX and nuclei followed by microscopy imaging and analysis. The number of γH2AX foci per nucleus is an accepted measure of the number of DNA double-strand breaks in single cells.
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