Phosphorylation of the H2AX protein is an early step in the double strand break 
(DSB) repair pathway; therefore, phosphorylated histone (γH2AX) foci scoring is 
widely used as a measure for DSBs. Foci scoring is performed either manually or 
semi-automatically using hand-operated capturing and image analysis software. In 
general, both techniques are laborious and prone to artifacts associated with 
manual scoring. While a few fully automated methods have been described in the 
literature, none of them have been used to quantify γH2AX foci in combination 
with a cell cycle phase analysis. Adding this feature to a rapid automated γH2AX 
foci quantification method would reduce the scoring uncertainty that arises from 
the variations in the background level of the γH2AX signal throughout the cell 
cycle. The method was set up to measure DNA damage induced in human mammary 
epithelial cells by irradiation under a mammogram device. We adapted a FISH 
(fluorescent in situ hybridization) Spot-counting system, which has a slide 
loader with automatic scanning and cell capture system throughout the thickness 
of each cell (z-stack), to meet our assay requirements. While scanning the 
sample, the system classifies the selected nuclei according to the signal 
patterns previously described by the user. For our purposes, a double staining 
immunofluorescence was carried out with antibodies to detect γH2AX and 
pericentrin, an integral component of the centrosome. We could thus distinguish 
both the number of γH2AX foci per cell and the cell cycle phase. Furthermore, 
restrictive settings of the program classifier reduced the "touching nuclei" 
problem described in other image analysis software. The automated scoring was 
faster than and as sensitive as its manually performed counterpart. This system 
is a reliable tool for γH2AX radio-induced foci counting and provides essential 
information about the cell cycle stage. It thus offers a more complete and rapid 
assessment of DNA damage.