Enhanced γ-H2AX DNA damage foci detection using multimagnification and extended depth of field in imaging flow cytometry

Abstract

Accurate and rapid methods for the detection of DNA damage foci in eukaryotic cells are central to DNA repair studies, which identify differences in DNA repair capacity in cell lines. Such assays have been important in delineating mechanisms of DNA repair in human cells. Previously we were the first to demonstrate the use of imaging flow cytometry for the detection of γ-H2AX foci in cells exposed to ionizing radiation causing the induction of DNA strand breaks. In this report we extend these studies and show an enhancement of foci quantitation and image resolution using next generation imaging flow cytometry with the Amnis Imagestream^X Mark II. We demonstrate using cell lines derived from normal individuals, and DNA double strand break repair defective cells that the number of foci observed is significantly increased when using 60× as compared to 40× magnification. Also, foci numbers and resolution is further increased with the application of the focus stacking (Extended Depth of Field-EDF) capacity activated. This report represents the first such demonstration of multimagnification and EDF for the enhanced quantitation of DNA damage in cells and provides a level of resolution, which near matches in situ microscopy methods for the detection of γ-H2AX foci.