High-Throughput γ-H2AX Assay Using Imaging Flow Cytometry.

The γ-H2AX assay is a sensitive and reliable method to evaluate radiation-induced DNA double-strand breaks. The conventional γ-H2AX assay detects individual nuclear foci manually, but is labor-intensive and time-consuming, and hence unsuitable for high-throughput screening in cases of large-scale radiation accidents. We have developed a high-throughput γ-H2AX assay using imaging flow cytometry. This method comprises (1) sample preparation from small volumes of blood in the Matrix™ 96-tube format, (2) automated image acquisition of cells stained with immunofluorescence-labeled γ-H2AX using ImageStream®, and (3) quantification of γ-H2AX levels and batch processing using the Image Data Exploration and Analysis Software (IDEAS®). This enables the rapid analysis of γ-H2AX levels in several thousand of cells from a small volume of blood with accurate and reliable quantitative measurements for γ-H2AX foci and mean fluorescence levels. This high-throughput γ-H2AX assay could be a useful tool not only for radiation biodosimetry in mass casualty events, but also for large-scale molecular epidemiological studies and individualized radiotherapy.