Validation of a flow cytometry-based detection of γ-H2AX, to
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. At present, flow cytometry is the most rapid method for detection of DSBs and cell viability. In this chapter, we provide our experience and methodological modification of flow cytometry protocol for the detection of γ-H2AX, a well-known marker of DSBs, in fixed mammalian fibroblasts.
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. Shigeaki Sunada, Hirokazu Hirakawa, Akira Fujimori, Mitsuru Uesaka, Ryuichi Okayasu; Oxygen Enhancement Ratio in Radiation-Induced Initial DSBs by an Optimized Flow Cytometry-based Gamma-H2AX Analysis in A549 Human Cancer Cells. Simple Western: gamma H2AX [p Ser139] Antibody (3F2) [NB100-74435] - Electropherogram image(s) of corresponding Simple Western lane view. Gamma H2AX antibody was used at 10 ug/ml dilution on Jurkat lysate(s). Rabbit polyclonal gamma H2A.X (phospho S139) antibody. Validated in WB, IHC, ICC/IF and tested in Mouse, Human.
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Additional reported applications (for the relevant formats of this clone) include: immunohistochemistry on paraffin embedded sections 2, immunofluorescence microscopy 3-9, Western blotting 10-12, and flow cytometry 1,13. Clone 2F3 cross-reacts with mouse 4. Intracellular staining protocol for Anti-H2A.X-Phosphorylated (Ser139) Antibody for Flow Furthermore, flow cytometric analysis of T2609 resulted in a better representation of fast repair kinetics than analysis of gamma-H2AX.
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The best sensitivity is achieved by computer analysis.
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. 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.
© 2016 2 Mar 2018 Using flow cytometry, we show here that phosphorylation at T2609 is faster in response to DSBs than gamma-H2AX. Furthermore, flow 27 Jun 2006 (a) Overlay of unirradiated (filled histogram) and UV-irradiated (open histogram) cells represents flow cytometry data that demonstrate increases 23 Mar 2006 methods, foci numbering, flow cytometry or Western blotting. the relationship between DSBs repair and γ–H2AX disappearance is not clear. H2AX phosphorylation at the SQ motif (γ-H2AX) has been Furthermore, by using DIM, flow cytometry, immunoblotting, and quantitative imaging microscopy 25 Jul 2017 γ-rays. DSB were enumerated with γH2AX foci using imaging flow cytometry. Phosphorylated form of histone H2AX (γH2AX) is a generally accepted UCB MNC were irradiated with γ-rays (0, 5, 10, and 50 cGy) and then& Clone REA502 recognizes the human and mouse histone H2AX antigen phosphorylated at serine 139 (pS139). X, γ-H2AX, γH2AX Intracellular flow cytometry (3) applications for H2AX pS139 Antibody, anti-human/mouse, REAfinity™.
20 Jun 2019 stress, both immunoblotting and flow cytometry analyses The γ-H2AX foci numbers per cell from 20-60 cells in each repeat of 3 biological
63 products gamma H2AX [p Ser139] Antibody · Applications: WB, FCM, ICC, IF, IHC, IHC-fr, IHC-p, ChIP · Reactivity: Hu, Ms, Rt, Ca · Conjugate/Tag: Unconjugated. 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. 70.00.
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At present, flow cytometry is the most rapid method for detection of DSBs and cell viability. In this chapter, we provide our experience and methodological modification of flow cytometry protocol for the detection of γ-H2AX, a well-known marker of DSBs, in fixed mammalian fibroblasts. The purpose of the present study was to assess immediate DNA damage after exposure to low level of ionizing radiation by the flow cytometric method of gamma-H2AX. Here, measurements of γ-H2AX immunofluorescence by microscopy and flow cytometry were compared as rapid biodosimetric tools for whole and partial body exposures. View This Abstract Online; Flow cytometric detection of gamma-H2AX to evaluate DNA damage by low dose diagnostic irradiation. Med Hypotheses. 2018; 115:22-28 (ISSN: 1532-2777) Gamma H2AX (gammaH2AX) is the phosphorylated version of histone H2AX and is a marker for double-stranded breaks (DSBs) caused by DNA damage (1-4).
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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.
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At present, flow cytometry is the most rapid method for detection of DSBs and cell viability.
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assays for the sensitive measurement of g-H2AX, using both microscopy and ﬂow cytometry (21–25). Analysis of g-H2AX by microscopy is still considered to be the most sensitive detection method and may discriminate between differential g-H2AX responses with respect to drug type and cell population makeup (7). However, the In contrast, flow cytometry allows simple detection of gamma-H2AX in a large number of cells . Several reports show that the level of gamma-H2AX as detected by flow cytometry correlates well with the number of DNA strand breaks, to the level of cell death and radiosensitivity ( 32–34 ). At present, flow cytometry is the most rapid method for detection of DSBs and cell viability. In this chapter, we provide our experience and methodological modification of flow cytometry protocol for the detection of γ-H2AX, a well-known marker of DSBs, in fixed mammalian fibroblasts.
The exposure to multiple CT scans causes more double strand breaks as compared to single scan. DNA damage can be studied by flow cytometric analysis of gamma-H2AX in human peripheral lymphocytes. H2AX phosphorylation was analyzed by flow cytometry analysis as previously described 23, with small modifications. After treatment, 1 mL of 0.1% BSA‐PBS was added to the samples and PBMCs were pelleted (5 min at 2000 g) followed by fixation in 0.25% paraformaldehyde‐PBS (8 × 10 6 cells/mL), for 10 min on ice. At present, flow cytometry is the most rapid method for detection of DSBs and cell viability.