The fixed end-point assay is based on fluorescent staining and immunofluorescence detection of cells grown on standard high-density microplates. Specific fluorescent probes effectively measure differences between treated and nontreated cells with respect to phospho-H2AX (Ser139) activity and nuclear localization. Phosphorylated human H2AX is detected with a specific primary antibody and a secondary antibody conjugated to DyLight* 549 Fluor. Hoechst dye provides identification of cell number, DNA content and nuclear morphology to enable characterization of target distribution. The procedure was optimized with the ArrayScan* HCS Reader and Compartmental Analysis BioApplication Software to provide automated plate handling, focusing, cell image acquisition, analysis and quantification of H2AX activation. Induction of DNA damage by treatment with compounds such as etoposide leads to phosphorylation of H2AX. The output parameter for this assay is the nuclear intensity of phosphor-H2AX staining after treatment. Cells labeled using this kit also can be imaged by fluorescence or confocal microscopy.
Required Accessories: Fluorescence microscope, Thermo Scientific Cellomics ArrayScan* VTI or other HCS reader
The nucleosome is made of four core histone proteins (H2A, H2B, H3 and H4). H2AX belongs to a H2A family of histones. DNA damage induction by various agents leads to rapid phosphorylation of H2AX at Ser139 (also known as gamma H2AX) by ATM, ATR or DNA protein kinase leading to the formation of DNA foci at the site of DNA double-strand breaks (DSBs). Phosphorylated H2AX helps in recruiting the proteins responsible for double-strand break repair.