Automated High-Content Screening of γ-H2AX Expression in HeLa Cells

Due to their inherent nature, DNA strands can be easily broken by various environmental factors including chemical agents and ionizing radiation. Unrepaired DNA double-stranded breaks (DSBs) may result in genetic instability and have a strong negative impact on the integrity of the genome. It has been found that DSBs are always followed by phosphorylation of histone protein H2AX, a member of the H2A family, and immunocytochemical detection of phosphorylated H2AX (referred to as γ-H2AX) is one of the frequently used techniques for assessing DNA damage. Usually such an assessment is done manually under the microscope which is not practical for analyzing large numbers of cells and prevents researchers from rapid and unbiased testing of novel drug compounds. To solve this problem we attempted to do automated assessment of DSBs by using a High-Content Screening (HCS) platform. As a result of this effort, we developed an easy to run HCS protocol for accurate analysis of DSBs in HeLa cells treated with camptothecin as a model. By varying the time of camptothecin treatment and its concentration we were able to study the dynamics of DSBs and perform a statistical analysis.Results of our study indicate that DSBs can be investigated using a HCS platform that enable the analysis of large numbers of experimental data points in a fast and a highly accurate manner. The protocol presented in this chapter can be easily adapted for screening libraries containing substantial numbers of chemical compounds for their efficiency to induce or/and repair DNA breaks.