Validation of a flow cytometry-based detection of γ-H2AX, to measure DNA damage for clinical applications. Artikel i vetenskaplig tidskrift, refereegranskad.

Histone H2A.X (H2AX) is a member of the histone H2A family which is one of the four core histones making up the nucleosome core particle. In eukaryotes, DNA double strand breaks (DSBs) have been shown to trigger the phosphorylation of serine 139 at the carboxy terminus of histone H2AX resulting in gamma-H2AX.

Bourton et al., 2011 have recently demonstrated using nonimaging flow cytometry, that in peripheral blood lymphocytes (PBL) derived from radiotherapy patients that experienced severe acute and delayed normal tissue toxicity, there was a persistence of γ-H2AX foci following exposure to 2 Gy gamma radiation. Flow Cytometry, Methanol Permeabilization Protocol for Rabbit Antibodies A. Solutions and Reagents. All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Methanol) #13593, or individually using the catalog numbers listed below. pubmed.ncbi.nlm.nih.gov tissues. gamma-H2AX has already been investigated in a variety of cancer types, including breast, lung, colon, cervix, and ovary cancers. The prognostic value of gamma-H2AX is indicated in certain cancer types, such as breast or endometrial cancer, but further investigation is needed to establish gamma-H2AX as a prognostic marker. Phosphorylated H2AX (also termed, gamma-H2AX) functions to recruit and localize DNA repair proteins or cell cycle checkpoint factors to the DNA-damaged sites.

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The best sensitivity is achieved by computer analysis. Using immunofluorescence and imaging flow cytometry, foci were measured in untreated cells and at 0.5, 3, 5 and 24 hours post-irradiation. In all lymphoblastoid cells treated with 2 Gy gamma radiation, there was a predictable induction of DNA strand breaks, with a modest but significant retention of foci over 24 hours in irradiated cells treated with Olaparib (ANOVA P 0.05). flow cytometry (1) immunocytochemistry (2) ELISA (2) dot blot (1) Host Species. mouse (1) Species Reactivity. Anti-Gamma H2AX (phospho-Ser139) antibody, Rabbit H2AX, H2a.x, H2afx, H2A/X 89 citations have been found for this product All applications Flow cytometry/Cell sorting (FC/FACS) Immunocytochemistry (ICC) Immunocytochemistry-immunoflourescence (ICC-IF) Immunofluorescence (IF) Immunohistochemistry (IHC) Immunohistochemistry-immunofluorescence (IHC-IF) Immunohistochemistry-paraffin (IHC-P) Immunoprecipitation (IP) Western Blotting (WB) Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] - BSA and Azide free Abcam catalog: ab215967 BioAssay record AID 1254631 submitted by ChEMBL: Induction of DNA damage in human HCT116 cells at G0/G1 phase at 30 uM after 24 hrs by gamma-H2AX-staining based-flow cytometry.

This process is believed to play a key role in the repair of DNA damage. In this study, we established a flow cytometry (FCM) system for measuring radiation-induced phosphorylated histone H2AX (gammaH2AX) in cultured human T lymphocytes to evaluate individual radiation sensitivity in vitro.

In this way, phosphorylated H2AX promotes DNA repair and maintains genomic stability and thus helps prevent oncogenic transformations. To this end, we selected 14 well-known genotoxic compounds and compared them with 10 nongenotoxic chemicals, using CHO-9 cells because they are well characterized as to DNA repair and DDR. We quantified gamma-H2AX foci manually and automatically.

26 Apr 2010 flow cytometry on a FACScan station with CellQuest software using the FL1 for FITC labeled caspase-3 active form or γ-H2AX. Western blotting.

Artikel i vetenskaplig tidskrift, refereegranskad.

Lastly, flow cytometry has been used to analyze γ-H2AX; however, flow cytometry methods are not readily integrated into HTS. Although each of the aforementioned methods of evaluating γ-H2AX is effective and has provided important information, there is still a need for an analytical high throughput assay that is capable of screening radiomodifying drugs across diverse cell lines and in vivo 2011-09-23 · Here, measurements of γ-H2AX immunofluorescence by microscopy and flow cytometry were compared as rapid biodosimetric tools for whole and partial body exposures. Ex vivo uniformly X-irradiated blood lymphocytes from one donor were used to generate a universal biexponential calibration function for γ-H2AX foci/intensity yields per unit dose for time points up to 96 hours post exposure. 2010-02-04 · Gamma-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin. Nucleic Acids Res 2008; 36 : 5678–5694. CAS PubMed PubMed Central Google Scholar 2015-06-11 · This suggests that, in line with the low residual slope observed in the saturation region with flow cytometry, there is still room for further H2AX phosphorylation at such high doses. These results are further supported by the direct comparison of flow cytometry data and integral fluorescence intensity as extracted from microscopy pictures which is shown in S1 Fig .
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P-H2AX flow cytometry assay in the clinic in a controlled manner. Finally, using immunostaining in optimized method for measurement of gamma-H2AX in. H2AX (gH2AX) was performed by flow cytometry to assess DNA repair defects in a FI Ratio γ. H2AX. 60.00.

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This process is believed to play a key role in the repair of DNA damage. In this study, we established a flow cytometry (FCM) system for measuring radiation-induced phosphorylated histone H2AX (gammaH2AX) in cultured human T lymphocytes to evaluate individual radiation sensitivity in vitro.

Using flow cytometry, we show here that phosphorylation at T2609 is faster in response to DSBs than gamma-H2AX. Furthermore, flow cytometric analysis of  Quantification of gamma-H2AX foci can be achieved by various methods such as Western blotting, flow cytometry, visual analysis and computational analysis  The advantage of flow cytometric analysis is that DSB formation and repair can be studied in relationship to cell cycle phase or expression of other proteins. However, γ-H2AX is not able to monitor repair kinetics within the first 60 min postirradiation, a period when most DSBs undergo repair. Öppna fliken parametrar i fönstret cytometer och välj parametrarna enligt tabell 1. Vi tackar Flow Cytometry Facility team på tyska cancer Research Center validation for radiation-induced gamma-H2AX foci in human cells. Att kvantifiera procentandelen av celler som har γ-H2AX-positiva foci, räkna varje cell Att kvantifiera antalet av γ-H2AX foci, räkna γ-H2AX foci per cell.