Typically, cells are fixed with formaldehyde to stabilize the cell membrane, and then permeabilized with detergent or. Flow cytometry protocols flow cytometry guide biorad. This purified mab is highly specific for phosphohistone h2a. Immunofluorescent staining and bioimaging analysis of cultured cells can be. H2ax staining can be performed with whole blood smears or isolated leukocytes using standard protocols for blood processing and immunofluo rescence. Does anyone have a protocol for gamma h2ax analysis using the. Histone h2ax is a 14 kda ubiquitous member of the h2a histone family that contains an evolutionary conserved sq motif at the cterminus in eukaryotes. A sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument. Rabbit polyclonal antigamma h2ax p ser9 antibody dna doublestrand break marker cited in 90 publications.
A modification of the basic immunofluorescent staining and flow cytometric analysis protocol can be used for the simultaneous analysis of surface molecules and intracellular antigens at the singlecell level by flow cytometry. Flow cytometry is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles. H2ax immunofluorescence for the detection of tissue. H2ax detection utilize laborious and lowthroughput microscopybased methods. H2ax also termed, gammah2ax functions to recruit and localize dna repair. Preparation and storage store undiluted at 4c and protected from prolonged exposure to light. H2ax foci levels in cells provides a sensitive and reliable method for quantitation of the radiationinduced dna damage response.
Manual differential count of 200 cells after maygrunwald stain showed. The majority of flow cytometers are now digital systems. It was found that the gammah2ax signal only increased 23fold in clmtreated wbc using published protocols. For low dose group blood was collected within 1 h after exposure in edta vaccutainers and immediately kept on ice. H2ax is abundant, fast, and correlates well with each dsb, it is the most sensitive marker that can be used to examine the dna damage produced and the subsequent repair of the dna lesion.
H2ax is a sensitive marker for dna doublestrand breaks dsbs, but the variability of h2ax expression in different cell and tissue types makes it difficult to interpret the meaning of the. The sample is focused to ideally flow one cell at a time through a laser beam and the light. My problem is even my control cells nonirradiated showing many foci some cells more than 10. Western blot and flow cytometry techniques may also be informative pilch et al. The objective of the present study was to develop a rapid, highthroughput.
This is one of the simplest and most common staining methods, where live or fixed cells are incubated with directly labeled antibodies against cell surface antigens. Serine 9 within this motif becomes rapidly phosphorylated to yield a form known as. Wb, simple western, flow, iccif, ihc, ihcfr, ihcp, chip, ko. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.
Cytometric assessment of histone h2ax phosphorylation. This manual method is, however, labor intensive and will be difficult to adapt. Cytometric assessment of histone h2ax phosphorylation ncbi. H2ax response in mice and found that even minor impairments in dsb repair lead to excessive dna damage accumulation during fractionated. Measurement of h2ax with the use of multiparameter flow or laser scanning cytometry seems to be particular advantageous as h2ax phosphorylation can be determined in individual cells with high sensitivity and accuracy and h2ax expression in cell populations can be correlated with dna content or induction of apoptosis 79, 80. For our purposes, a double staining immunofluorescence was carried out with antibodies. The flow cytometry protocols below provide detailed procedures for the treatment and staining of cells prior to using a flow cytometer. H2ax phosphorylation was analyzed by flow cytometry analysis as previously described 23, with small modifications. X phospho s9 antibody 9f3 ab26350 overlay histogram showing hela cells stained with ab26350 red line. X ser9, clone jbw301 is a well published mouse monoclonal antibody validated in chip, icc, if, wb. Gammah2axbased dose estimation for whole and partial body. H2ax, a wellknown marker of dsbs, in fixed mammalian fibroblasts. Can you help me for first and second antibody for gamma h2ax analysis using flow cytometry.
Measuring dna damage and repair in mouse splenocytes after. Does anyone have a protocol for gamma h2ax analysis using the flow cytometry. Combining the strength of flow cytometry and conventional microscopy. Combine the recommended quantity of each primary antibody in an appropriate volume of flow cytometry staining buffer so that the final staining volume is 100 l i.
The apoptosis, dna damage, and cell proliferation kit contains key markers for the simultaneous determination of these important cellular states by flow cytometry saving time and samples, and improving results. Pbs was added to the samples and pbmcs were pelleted 5 min at 2000 g followed by fixation in 0. H2ax antibody, antiphosphohistone h2ax pser9 antibody produced in rabbit. Lymphocytes were isolated and were fixed in 80% chilled ethanol and stored at. H2ax and rad51 dna repair biomarkers in dna repair normal cell lines mrc5sv1 and nb1htert, an atmdeficient cell line at5biva derived from an ataxia. H2ax staining, which can be performed on tissue sections, promises to overcome those limitations. A gamma h2ax kit and antibody allows the assessment of dna damage and dna repair for elisa based assays, immunohistochemistry or flow cytometry. Antiphosphohistone h2ax pser9 antibody h2ax antibody. Apoptosis, dna damage, and cell proliferation kit subject. H2ax if could be a useful tool to extend this genotoxicity test panel, a comparison with the current gold standard method the comet assay was conducted.
Intensity of cellular green fitc and red pi fluorescence is measured by flow cytometry and bivariate analysis of the data is used to correlate the presence of dsbs with the cell cycle phase. This is the job of the signal processing electronics. Jun 26, 2008 the stained nuclei can be analyzed by flow cytometry to monitor the level of gamma h2ax to determine the level of dsbs and dna content and to determine the cell cycle stage. The use of fluorescence measurements can be extended to the detection of total g h2ax protein levels. Estimation of gamma h2ax can be done either by immunofluorescence or by flow cytometry, the immunofluorescence technique has been used extensively and effectively to study the dna damage caused by ionizing radiation wherein gamma h2ax has emerged as a sensitive biomarker for dna damage detection. Mouse fibroblast cell line transfected with human gene. Problem with gamma h2ax foci staining protocol online. Does anyone have a protocol for gamma h2ax analysis using.
Flow cytometric detection of gammah2ax to evaluate dna damage by. In the detection of genomic damage, gamma h2ax is especially important in research involving cancer treatment and therapy, and environmental biomonitoring. Jul 15, 2017 at present, flow cytometry is the most rapid method for detection of dsbs and cell viability. H2ax protocol was optimized for small volumes 100 l of blood in matrix. My interest is to see the effect of my gene on dna damage after irradiation 5 gy. Here we describe the use of c h2ax andor 53bp1 immunofluorescence microscopy and c h2ax flow cytometry as quantitative biomarkers for whole and partial body exposure to ionising radiation. The h2ax assay was done and 10,000 cells were analysed for gamma h2ax positivity in flowcytometer. Flow cytometry was performed on a bd lsr ii system. Staining for gammah2ax was done by adding 5000100 000 cells to 150200. Proceed to step 8 for purified or biotinylated primary antibodies.
H2ax can be detected by immunoblotting and immunostaining using microscopic or flow cytometric detection. Flow or laser scanning cytometry offers the possibility to rapidly quantify. H2ax, as measured by flow cytometry, could be used as an indicator of tissue radiosensitivity. The fitc signal was measured in isolated cells using flow cytometry. An optimized method for detecting gammah2ax in blood cells. In this chapter, we provide our experience and methodological modification of flow cytometry protocol for the detection of. Antitumor drugs, cell cycle phase, dna damage, doublestrand dna breaks, flow cytometry, histone h2ax phosphorylation, immunofluorescence. Feb 06, 2007 flow cytometry measurement for gammah2ax. Histone h2ax antibody staining protocol for immunohistochemistry. H2ax as protein biomarker for radiation exposure iss. The cells were fixed with 80% methanol 5 min and then permeabilized with 0. Additional reported applications for the relevant formats of this clone include. At present, flow cytometry is the most rapid method for detection of dsbs and cell viability. The stained nuclei can be analyzed by flow cytometry to monitor the level of gammah2ax to determine the level of dsbs and dna content and to determine the cell cycle stage.
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