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

By Younghyun Lee, Qi Wang, Igor Shuryak, David J Brenner, Helen C. Turner

Posted 15 May 2019
bioRxiv DOI: 10.1101/637371 (published DOI: 10.1186/s13014-019-1344-7)

Background: Measurement of γ-H2AX foci formation 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 MKII) platform to evaluate DNA double strand break (DSB) repair kinetics in human peripheral blood cells after exposure to ionizing irradiation. Methods: The γ-H2AX protocol was optimized for small volumes (100 µl) of blood in Matrix™ 96-tube format and blood cell lymphocytes were identified and captured by ISX INSPIRE™ software and analyzed by Data Exploration and Analysis Software. Results: Presented here are: 1) dose response curves based on γ-H2AX fluorescence intensity and foci number, 2) measurements of DNA repair kinetics up to 24 h after exposure to 4 Gy γ rays and, 3) a mathematical approach for modeling DNA DSB rejoining kinetics using two key parameters a) rate of γ-H2AX decay, and b) yield of residual unrepaired breaks. Conclusions: The results indicate that the IFC-based γ-H2AX protocol may provide a practical, high-throughput and inexpensive platform for measurements of individual global DSB repair capacity and facilitate the prediction of precision medicine concepts.

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