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An optimized method for detecting gamma-H2AX in blood

Se hela listan på academic.oup.com 2012-12-31 · The γ-H2AX assay has also been used to monitor formation and persistence of DNA damage and cytotoxicity in human cancer cells produced by another Auger emitter, 111 In, that has been delivered to cell nuclei by pharmaceuticals that target human epidermal growth factor receptor (EGFR)-positive cancers and HER2/neu-amplified breast cancers , . PDF | On Jan 1, 2008, I.M. Rakiman and others published γ-H2AX assay: a technique to quantify DNA double strand breaks | Find, read and cite all the research you need on ResearchGate Gamma-H2AX assay is an immuno-fluorescence experiment that enables detecting the location and number of DNA double strand breaks (DSBs) in cells. Assuming that a single gamma-H2AX focus corresponds to a single DSB, one can convey the severity of cellular damage by the number of gamma-H2AX foci per cell (FPC) [1]. Thus, with γH2AX we have a bio-indicator on hand for critical DNA lesions and severe genomic replication stress. Hence, the γH2AX assay is not an assay for detecting any type of DNA damage, but for biologically relevant and severe DNA damage (including DNA interstrand crosslinks that give rise to DSBs; Nikolova et al., 2012). Phosphorylated H2AX (gamma-H2AX) is essential to the efficient recognition and (or) repair of DNA double strand breaks (DSBs), and many molecules, often thousands, of H2AX become rapidly phosphorylated at the site of each nascent DSB. An antibody to gamma-H2AX reveals that this highly amplified process generates nuclear foci. 2017-02-03 · Phosphorylated H2AX (γ-H2AX) is a sensitive marker for DNA double-strand breaks (DSBs), but the variability of H2AX expression in different cell and tissue types makes it difficult to interpret the meaning of the γ-H2AX level.

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Following radiation exposure, histone H2AX is rapidly phosphorylated by ATM and/or DNA-PK kinases at or near the vicinity of DNA DSB sites to form γ-H2AX [ 14 ]. Thus, with γH2AX we have a bio-indicator on hand for critical DNA lesions and severe genomic replication stress. Hence, the γH2AX assay is not an assay for detecting any type of DNA damage, but for biologically relevant and severe DNA damage (including DNA interstrand crosslinks that give rise to DSBs; Nikolova et al., 2012). “Neutral” lesions, such as N7-methylguanine, which does not block replication, will likely not be detected.

An assay for γH2AX generally reflects the presence of double-strand breaks in DNA, though the assay may indicate other minor phenomena as well. 2018-06-01 · Gamma-H2AX assay has proved useful for detecting DNA damage at doses of radiation down to 1 mGy and it is said to be 100 times more sensitive than other methods , . Reportedly the gamma-H2AX can be quantified either by immunoflourescence or flow cytometry [14] .

An optimized method for detecting gamma-H2AX in blood

To determine the dose response and repair kinetics of gamma-H2AX ionizing radiation-induced foci in VH10 human fibroblasts exposed to mixed beams of 241Am alpha particles and X-rays. 2016-03-04 · The gammaH2AX assay for genotoxic and nongenotoxic agents: comparison of H2AX phosphorylation with cell death response. Toxicol Sci 140, 103–117 (2014).

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Histone H2AX: Products. Histone H2AX is one of a number of core histone proteins. In the cellular response to genotoxic insults, ATM and related protein kinases phosphorylate the carboxyl-terminal tail of the H2AX protein (gamma-H2AX). gamma-H2AX marks the site of damage and provides a nucleation site for the formation of damage response and repair complexes. This modification, called gamma-H2AX (phospho-Ser139), triggers cell cycle arrest and DNA damage response repair, which makes this assay particularly appropriate for studying genome stability, cell cycle, DNA repair, and more broadly as a biomarker for cancer and for aging process investigations. We quantified gamma-H2AX foci manually and automatically.

We quantified gamma-H2AX foci manually and automatically. In addition, total gamma-H2AX activation was determined by flow cytometry. For all chemicals the cytotoxic dose response was assayed by a metabolic cytotoxicity assay. Listed are ELISA Kits for the detection of H2AX, an alias name of H2A histone family member X. The human protein, encoded by the gene H2AFX, is 143 amino acid residues long and has a mass of 15,145 daltons. Evaluation of the Gamma-H2AX Assay for Radiation Biodosimetry in a Swine Model Maria Moroni 1,†, Daisuke Maeda 2,†, Mark H. Whitnall 1, William M. Bonner 2 and Christophe E. Redon 2,* 1 Armed Forces Radiobiology Research Institute, Uniformed Services University, Bethesda, MD 20889-5603, USA; E-Mails: maria.moroni@usuhs.edu (M.M.); Gamma-H2AX foci detection is the standard method to quantify DNA double-strand break (DSB) induction and repair.
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Use of the gammaH2AX assay for assessing the genotoxicity of polycyclic  DNA double-strand break repair kinetics in minipig blood lymphocytes and fibroblasts, based on the γ-H2AX assay, were similar to those observed in their  Keywords: DNA double-strand break (DSB), high-throughput assay, γ-H2AX, Phosphorylation of the histone protein H2AX to form γ-H2AX foci directly  ). The gamma-H2AX foci assay is a sensitive surrogate marker of radiation induced DNA double strand breaks (Ivashkevich et al. 2012  The focus of the study is an intercomparison of laboratories' dose-assessment performances using the y. H2AX foci assay as a diagnostic triage tool for rapid.

007954116. The sensitivity of the γ-H2AX assay is limited by the variability of foci levels in untreated cells, and is therefore cell type-dependent. Unirradiated pe- ripheral blood  1 Feb 2013 Experimental Design: γ-H2AX foci formation was validated preclinically in comparison with the Comet assay, and evaluated  21 Aug 2012 This assay is based on the phosphorylation of H2AX histone in response to Use of the [gamma]H2AX assay for assessing the genotoxicity of  The role of γ-H2AX foci in the DNA damage signaling pathway induced by For the alkaline comet assay, the comet assay method from Trevigen was used  29 Apr 2003 DSBs (Foci) Can Be Quantified After Doses as Low as 1 mGy. We next determined the sensitivity of the γ-H2AX assay with regard to the lowest IR  30 Sep 2016 Upon phosphorylation H2AX is referred to as gamma-H2AX. Given that only H2AX histones near the site of DNA damage are phosphorylated,  16 Apr 2013 An assay of γ-H2AX focus formation, a marker of DNA damage, after 5-FU treatment revealed good correlation with the levels of RRM-1 protein  28 Sep 2015 The γ-H2AX assay has a potential for triage despite the fact of limited stability. To conclude none of the assay could fulfil all the criteria of an  photo bleaching (FRAP assays), gene knockdown using RNAi technology, DNA damage evaluation in single cell by gamma-H2AX staining and Comet assays  xTAG Technology, combined with Luminex instruments, is a complete solution for mid-density arrays and performing multiplexed nucleic acid assays. Investigating γ H2AX as a Biomarker of Radiosensitivity Using Flow Cytometry subsets, CD19 in particular, might be proved to be a more sensitive assay.
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In this study, we investigated the induction and decay of γ-H2AX foci of different tumor cell lines and fibroblasts with known mutations in DNA damage repair genes, including ATM, LigIV, DNA-PKcs, Rad51 and Rad54. A radiation dose of 2.4 Gy was used for either an acute single high γ-H2AX Standard - The kit contains 20 μL of γ-H2AX Standard at a concentration of 1 μM. Centrifuge before opening vial and aliquot to avoid repeated freeze/thaw cycles. Add 3 μL of γ-H2AX Standard to 72 μL Assay Buffer to prepare 40 nM of γ-H2AX Standard (diluted 1:24).

Unirradiated pe- ripheral blood  1 Feb 2013 Experimental Design: γ-H2AX foci formation was validated preclinically in comparison with the Comet assay, and evaluated  21 Aug 2012 This assay is based on the phosphorylation of H2AX histone in response to Use of the [gamma]H2AX assay for assessing the genotoxicity of  The role of γ-H2AX foci in the DNA damage signaling pathway induced by For the alkaline comet assay, the comet assay method from Trevigen was used  29 Apr 2003 DSBs (Foci) Can Be Quantified After Doses as Low as 1 mGy. We next determined the sensitivity of the γ-H2AX assay with regard to the lowest IR  30 Sep 2016 Upon phosphorylation H2AX is referred to as gamma-H2AX.
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By Sandrine Roch-Lefèvre, Marco Valente, Philippe Voisin and Joan-Francesc Barquinero. Phosphorylation of histone H2AX to form gamma-H2AX is a known marker for irradiation-induced DNA DSBs. However, the first generation assay involves the  H2AX (also termed, gamma-H2AX) functions to recruit and localize DNA repair proteins and the Triton X-100 Perm/Wash protocols (see Recommended Assay . Musthafa A, Djuita F, Kurnia I. Expression of γ-H2AX using immunoflourescence assay as an adaptive response of PBMC in radiation workers at Dharmais. The gamma-H2AX assay enables screening for functional integrity of DNA double strand break (DSB) repair, and a broad range of genetic and epigenetic  20 products Compare H2AX ELISA Kits from leading suppliers on Biocompare. The ELISA ( enzyme-linked immunosorbent assay) is a widely used application for detecting and quantifying proteins and Human gamma h2ax ELISA Kit. Laboratories around the world now use the gamma-H2AX assay developed by Bonner to study how cells sense and respond to double-strand breaks. The CCR   H2AX dose-response curves in human lymphocytes exposed to ionizing radiation The gamma-H2AX assay – an effective alternative for the comet assay in  When the γ -H2AX foci which mark the DSBs are stained, individual breaks are detectible, making the assay suitable for situations requiring great sensitivity.