DNA DSBs present the most serious threat to the cell. These approaches have the potential to improve screening of compounds that either enhance DNA damage or protect against its deleterious effects.ĭNA damage in cells can be triggered by endogenous mechanisms, exogenous factors, or a combination of both, resulting in a multitude of alterations including DNA base modifications, single-strand breaks, and double-strand breaks (DSBs) 1. Lanthanide based immunodetection of γ-H2AX offers superior detection and a user-friendly workflow. Comparative bulk fluorescent signals achieved only micromolar sensitivity. Time resolved fluorescence detection of europium as a chelated complex enabled quantitative measurement of γ-H2AX foci with nanomolar resolution. Etoposide induced DNA damage in A549 carcinoma cells was compared across all test platforms. 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. The novelty of this work is the application of a time-resolved fluorescence assay using dissociation-enhanced lanthanide fluorescence immunoassay for quantitative measurements of γ-H2AX. Traditional γ-H2AX detection involves counting individual foci within individual nuclei. Phosphorylation of the histone protein H2AX to form γ-H2AX foci directly represents DNA double-strand break formation.
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