Abstract
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®X, 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.
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Acknowledgements
This study was supported by a grant of the Korea Institute of Radiological and Medical Sciences, funded by Nuclear Safety and Security Commission of the Republic of Korea (50091-2023). This work was also supported by the Center for High-Throughput Minimally-Invasive Radiation Biodosimetry, National Institute of Allergy and Infectious Diseases (grant number U19AI067773).
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Lee, Y., Wang, Q., Seong, K.M., Turner, H.C. (2023). High-Throughput γ-H2AX Assay Using Imaging Flow Cytometry. In: Barteneva, N.S., Vorobjev, I.A. (eds) Spectral and Imaging Cytometry. Methods in Molecular Biology, vol 2635. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3020-4_7
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DOI: https://doi.org/10.1007/978-1-0716-3020-4_7
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