| Autor: |
Felicite K. Noubissi, Xiaofei Wang, Sandra M. Davern, Larry J. Millet, Hannah G. Leppert, Amber A. McBride |
| Rok vydání: |
2021 |
| Předmět: |
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| DOI: |
10.21203/rs.3.rs-140475/v1 |
| Popis: |
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. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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