Imaging flow cytometry as a sensitive tool to detect low‐dose‐induced DNA damage by analyzing 53BP1 and γH2AX foci in human lymphocytes

Abstract

Ionizing radiation induced foci (IRIF) are considered the most sensitive indicator for DNA double-strand break (DSB) detection. Monitoring DSB induction by low doses of ionizing radiation is important due to the increasing exposure in the general population. cH2AX and 53BP1 are commonly used molecular markers for in situ IRIF assessment. Imaging flow cytometry (IFC) via ImageStream system provides a new opportunity in this field. We analyzed the formation of 53BP1, cH2AX foci and their co-localization induced by c-rays (2, 5, 10, 50, 200 cGy) in human lymphocytes using ImageStream and the automated microscopic system Metafer. We observed very similar sensitivity of both systems for the detection of endogenous and low-dose-induced IRIF. Statistically significant induction of cH2AX foci was found at doses of 2 and 10 cGy using ImageStream and Metafer, respectively. Statistically significant induction of 53BP1 foci was evident at doses !5 cGy when analyzed by IFC. Analysis of the colocalizing foci by ImageStream and Metafer showed statistical significance at doses !2 cGy, suggesting that foci co-localization is a sensitive parameter for DSB quantification. Assessment of cH2AX, 53BP1 foci and their co-localization by Metafer and ImageStream showed similar linear dose responses in the low-dose range up to 10 cGy, although IFC showed slightly better resolution for IRIF in this dose range. At higher doses, IFC underestimated IRIF numbers. Using the imaging ability of ImageStream, we introduced an optimized assay by gating cH2AX foci positive (with 1 or more cH2AX foci) and negative (cells without foci) cells. This assay resulted in statistically significant IRIF induction at doses ! 5cGy and a linear dose response up to 50 cGy. In conclusion, we provide evidence for the use of IFC as an accurate high throughput assay for the prompt detection and enumeration of endogenous and low-dose induced IRIF. V C 2015 International Society for Advancement of Cytometry Key terms imaging flow cytometry; ImageStream; Metafer; human lymphocytes; cH2AX; 53BP1; ionizing radiation DNA double strand breaks (DSB) are known as the most deleterious type of DNA damage induced by ionizing radiation and other genotoxic agents. Misrepaired or unrepaired DSB lead to genome rearrangements and contribute to carcinogenesis. Currently, the most sensitive and specific method for DSB analysis is enumeration of small repair centers formed at the locations of DSB. These centers are known as ionizing radiation induced foci (IRIF) or DNA repair foci. IRIF are dynamic structures containing thousands of copies of proteins involved in various aspects of DSB repair and DNA damage response. Phosphorylated histone H2AX variant (cH2AX) and p53 binding protein 1 (53BP1) are commonly accepted molecular markers of IRIF (1,2). Upon DSB induction, histone H2AX molecules become phosphorylated within

Low numbers of pre-leukemic fusion genes are frequently present in umbilical cord blood without affecting DNA damage response

“…Two independent approaches for measuring the level of endogenous DNA repair foci, namely fluorescence microscopy by Metafer system (Figure 2A) and imaging flow cytometry by ImageStream system (Figure 2B, 2C) were used and compared. In line with previously published data, some γH2AX and 53BP1 foci did not co-localize due to various kinetics of γH2AX and 53BP1 at the locations of DSB [29]. By analyzing γH2AX, 53BP1 foci and their co-localization using imaging flow cytometry we did not find significant difference in endogenous DSB between ten PFG + and ten PFG − samples ( p = 0.16, ANOVA) (Figure 3A).…”

“…As a positive control, we analyzed induction of γH2AX/53BP1 foci 30 min post-irradiation with γ-rays in the dose range of 2–50 cGy. We found statistically significant increase of γH2AX and 53BP1 foci already at 2 cGy (data not shown) confirming very high sensitivity of our assays [29]. …”

“…Cells from ten PFG + and ten matched PFG − probands were analyzed for endogenous DSB using imaging flow cytometry by ImageStream X-100 (Amnis, Seattle, USA) and fluorescence microscopy by the Metafer slide scanning system (MetaSystems, Altlusheim, Germany) as previously described [29]. For the ImageStream analysis, each sample was probed using either polyclonal γH2AX antibody (γH2AXp, Cell Signaling Technology Danvers, MA, USA) in combination with CD34 marker for HSPC (MACS Miltenyi Biotec, Bergisch Gladbach, Germany) or monoclonal γH2AX antibody (γH2AXm, Novus Biologicals, United Kingdom) with polyclonal 53BP1 antibody (Novus Biologicals, United Kingdom).…”

Extracellular Vesicles Mediate Radiation-Induced Systemic Bystander Signals in the Bone Marrow and Spleen

“…The fluorescent microscopy protocol is considered a more accurate and more specific method than evaluating the frequency of event-positive cells by flow cytometry (28). However, the latter method is much quicker, allows the quantification of much higher number of cells, and in this way, increases the statistical power in cases where the number of alterations is low (29). As expected, a dose-dependent increase of DNA damage was detected in directly irradiated animals.…”

Masks in imaging flow cytometry

“…For example, Jenner et al, [2] used a combination of an intracellular and spot counting masks to enumerate intracellular bacteria. In their study of radiation damage to lymphocytes, Durdik and coworkers [7] used custom masks which combined Spot, Peak, and Intensity features. Studies by McGrath et al and by Katz and colleagues also show excellent examples of using mask combinations [5,8].…”