In Vitro High-Throughput Genotoxicity Testing Using γH2AX Biomarker, Microscopy and Reproducible Automatic Image Analysis in ImageJ-A Pilot Study with Valinomycin

(1) Background: The detection of DNA double-strand breaks in vitro using the phosphorylated histone biomarker (gamma H2AX) is an increasingly popular method of measuring in vitro genotoxicity, as it is sensitive, specific and suitable for high-throughput analysis. The gamma H2AX response is either detected by flow cytometry or microscopy, the latter being more accessible. However, authors sparsely publish details, data, and workflows from overall fluorescence intensity quantification, which hinders the reproducibility. (2) Methods: We used valinomycin as a model genotoxin, two cell lines (HeLa and CHO-K1) and a commercial kit for gamma H2AX immunofluorescence detection. Bioimage analysis was performed using the open-source software ImageJ. Mean fluorescent values were measured using segmented nuclei from the DAPI channel and the results were expressed as the area-scaled relative fold change in gamma H2AX fluorescence over the control. Cytotoxicity is expressed as the relative area of the nuclei. We present the workflows, data, and scripts on GitHub. (3) Results: The outputs obtained by an introduced method are in accordance with expected results, i.e., valinomycin was genotoxic and cytotoxic to both cell lines used after 24 h of incubation. (4) Conclusions: The overall fluorescence intensity of gamma H2AX obtained from bioimage analysis appears to be a promising alternative to flow cytometry. Workflow, data, and script sharing are crucial for further improvement of the bioimage analysis methods.

Automated summary

This article presents a new method for measuring in vitro genotoxicity using the phosphorylated histone biomarker (gamma H2AX). The authors used flow cytometry and microscopy to detect the gamma H2AX response and performed bioimage analysis using the open-source software ImageJ. They provided workflows, data, and scripts for further improvement of bioimage analysis methods.