A High-Throughput, Time-Resolved Fluorescence Approach Increases γ-H2AX Detection

Abstract Phosphorylation of the histone protein H2AX to form γ-H2AX foci directly represents DNA double-strand break formation. Traditional γ-H2AX detection involves counting individual foci within individual nuclei. Here, we present the development of a sensitive high-throughput assay to quantify γ-H2AX using dissociation-enhanced lanthanide fluorescence immunoassay and time-resolved fluorescence. For comparison, standard fluorescence detection was employed and analyzed either by bulk fluorescent measurements or by direct foci counting using BioTek Spot Count algorithm and Gen 5 software. Etoposide induced DNA damage in A549 carcinoma cells was compared across all test platforms. Time resolved fluorescence detection of europium as a chelated complex enabled quantitative measurement of γ-H2AX foci with nanomolar resolution. Comparative bulk fluorescent signals achieved only micromolar sensitivity. Lanthanide based immunodetection of γ-H2AX offers superior detection and a user-friendly workflow. These high throughput approaches can potentially improve screening of compounds that either enhance DNA damage or protect against its deleterious effects.