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 ImagestreamX 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. © 2015 The Authors. Published by Wiley Periodicals Inc. on behalf of ISAC.

Condensin II mutation causes T-cell lymphoma through tissue-specific genome instability

“…Fluorescent and bright-field images were captured using an ISx MKII (Amnis) using a 60× objective. Following acquisition and compensation, the data were analyzed with IDEAS (Amnis) software using a modified version of published methods (Parris et al 2015) as follows. In addition to the automatically generated system image masks, three further masks were built.…”

Imaging Flow Cytometry to Assess Antigen‐Presenting‐Cell Function

“…Imaging flow cytometry is a method that combines aspects of traditional flow cytometry and fluorescence microscopy, resulting in a platform for high‐throughput microscopy. Imaging flow cytometry has provided a novel approach to assess a number of immunological processes that have traditionally been very difficult to study, including cellular interactions such as synapse formation (Ahmed, Friend, George, Barteneva, & Lieberman, ; Markey et al., ; Markey, Gartlan, Kuns, MacDonald, & Hill, ), phagocytosis (Gartlan et al., ; Smirnov, Solga, Lannigan, & Criss, ), infection (Baxter et al., ; Haridas, Ranjbar, Vorobjev, Goldfeld, & Barteneva, ; Khoury et al., ), cell signaling and nuclear translocation (George et al., ; O. Maguire, Collins, O'Loughlin, Miecznikowski, & Minderman, ; Orla Maguire, Tornatore, O'Loughlin, Venuto, & Minderman, ), antigen presentation (Koyama et al., ), the cell cycle and mitotic division (Filby et al., ), apoptosis and DNA repair/damage (George et al., ; Parris et al., ), cell death (George et al., ), autophagy (Leveque‐El Mouttie et al., ; Pugsley, ), and morphological changes (Grimwade, Fuller, & Erber, ). The key advantage of this method is that it allows capture of not only immunofluorescence data (as is usual for standard flow cytometry) but also the spatial localization of the antibody targets of interest (as is usual for microscopy), rendering it particularly useful for study of immune function.…”

An open-source solution for advanced imaging flow cytometry data analysis using machine learning

“…Most IFC-based assays take advantage of the technologies’ inherent ability to measure fluorescence signals with spatial context. Such assays include measuring nuclear translocation [7], mitochondrial localisation [8], co-localization assays using “similarity” features [9], calcium signalling at the organelle level [10], organelle inheritance during mitosis [11], cell cycle phases [12], receptor activity [13], asymmetric cell division [14], [15], [16], fission yeast cell cycle [17], dendritic cell morphology [18], autophagy [19], [20], detection of DNA damage foci [21], [22] and modelling intracellular infection [23]. Most, if not all, of these assays would not be possible using traditional flow cytometry (lack of spatial information) or conventional imaging techniques (low throughput and poor quantitation).…”