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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. 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. 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 approaches have the potential to improve screening of compounds that either enhance DNA damage or protect against its deleterious effects.
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Details
1 Jackson State University, Department of Biology, Jackson, USA (GRID:grid.257990.0) (ISNI:0000 0001 0671 8898)
2 Oak Ridge National Laboratory, Biosciences Division, Oak Ridge, USA (GRID:grid.135519.a) (ISNI:0000 0004 0446 2659)
3 University of Tennessee, Center for Environmental Biotechnology, Knoxville, USA (GRID:grid.411461.7) (ISNI:0000 0001 2315 1184)
4 Tennessee State University, Department of Biological Sciences, Nashville, USA (GRID:grid.280741.8) (ISNI:0000 0001 2284 9820)
5 Oak Ridge National Laboratory, Radioisotope Science and Technology Division, Oak Ridge, USA (GRID:grid.135519.a) (ISNI:0000 0004 0446 2659)