Development of a high-throughput γ-H2AX assay based on imaging flow cytometry

Abstract

Measurement of γ-H2AX foci levels in cells provides a sensitive and reliable method for quantitation of the radiation-induced DNA damage response. The objective of the present study was to develop a rapid, high-throughput γ-H2AX assay based on imaging flow cytometry (IFC) using the ImageStream®X Mk II (ISX) platform to evaluate DNA double strand break (DSB) repair kinetics in human peripheral blood cells after exposure to ionizing irradiation. The γ-H2AX protocol was developed and optimized for small volumes (100 μL) of human blood in Matrix™ 96-tube format. Blood cell lymphocytes were identified and captured by ISX INSPIRE™ software and analyzed by Data Exploration and Analysis Software. Dose- and time-dependent γ-H2AX levels corresponding to radiation exposure were measured at various time points over 24 h using the IFC system. γ-H2AX fluorescence intensity at 1 h after exposure, increased linearly with increasing radiation dose (R2 = 0.98) for the four human donors tested, whereas the dose response for the mean number of γ-H2AX foci/cell was not as robust (R2 = 0.81). Radiation-induced γ-H2AX levels rapidly increased within 30 min and reached a maximum by ~ 1 h, after which time there was fast decline by 6 h, followed by a much slower rate of disappearance up to 24 h. A mathematical approach for quantifying DNA repair kinetics using the rate of γ-H2AX decay (decay constant, Kdec), and yield of residual unrepaired breaks (Fres) demonstrated differences in individual repair capacity between the healthy donors. The results indicate that the IFC-based γ-H2AX protocol may provide a practical and high-throughput platform for measurements of individual global DNA DSB repair capacity which can facilitate precision medicine by predicting individual radiosensitivity and risk of developing adverse effects related to radiotherapy treatment.