This algorithm and its applications for cell nucleus and foci detection will be described. H2ax foci in cell lines derived from patients with severe or mild mutations in a core non. Computational analysis of the number, area and density of gamma h2ax foci in breast cancer cells exposed to 111 indtpahegf or gammarays using image j software. The numbers of ld per cell and size were quantified by using an automatic measurement program of the imagej software. Count the number of nuclei in a field this is relatively easy as nuclei tend to be fairly well separated, similar in size and brightly stained. Since irradiations as well as image capture were carried out perpendicularly to the glass slide to which cells were attached, one particle traversal was visualized as one. H2ax assay to monitor dna damage and repair in translational cancer research. H2ax, 53bp1 and rad51 protein foci changes in mesenchymal. We used imagej as an established platform, as it is an image processing software that is routinely used by many investigators to analyze western.
Phosphorylation of the chromatin protein h2ax forming. H2ax is a known marker for irradiationinduced dna dsbs. Opens a permanent window frame within imagej and allows selection of different images. In radiation biology and molecular radiation oncology, dna damage and dna repair kinetics upon ionizing radiation ir are evaluated by counting protein clusters or accumulations of phosphorylated proteins. Gamma h2ax foci were quantified by using imagej software in the following sequence. Imagej is highly extensible, with thousands of plugins and scripts for performing a wide variety of tasks, and a large user community. H2ax foci by drugs readily detected with this assay.
Histone h2ax plays a crucial role in molecular and cellular responses to dna damage and in the maintenance of genome stability. The area of each individual foci was reported and averaged per image. Count nuclear foci imagej duke light microscopy core. The current standard to quantitate foci in opensource software is to. Can someone please tell me step by step on how to do this. Quantification of the fraction of dna in eu or heterochromatin conformation was performed with image j software using an intensity threshold algorithm set at 50% of the maximum signal from. H2ax fluorescence assay became one of the methods of choice for the detection of radiation induced dna double strand breaks dsb, after it was shown in 1998 by rogakou and coworkers that the h2ax histones are locally phosphorylated on serine 9 in presence of a dsb, giving rise to the socalled. Robotic specimen preparation with automated image acquisition and analysis can be used to quantify. H2ax foci was found to be affected by the initial radiation exposure with a smaller number of foci induced by subsequent exposures. Any software to count gamma h2ax s9 and patm foci in. H2ax antibodies are available, the dsbs can be visualized as discrete foci.
Interestingly, rad51 foci were not produced in irradiated resting cells either and this lack of foci was found for all doses examined figure figure4b. Any software to count gamma h2ax s9 and patm foci in dna damaged cells. Tufuse pro is a windows image fusion program that can be used to create extended depth of field focus blended images, extended exposure range exposure blended images, and other types of composite images. H2ax senses dsbs through rapid serine 9 phosphorylation, concurrently leading to. It runs the findfoci plugin to perform the processing work. Linking gamma h2ax foci and cancer in rat skin exposed to heavy ions and electron radiation. The background flattening size, or rolling ball diameter, was also set to 2 m as was seen in the image preprocessing step, to aid in accurate identification of each foci. It is downstream of ataxia telangiectasia mutated atm damage signaling pathway and there is an emerging role of the transcription factor foxo3a, a regulator of a variety of other pathways, in activating this signaling. I am starting to do some gamma h2ax focci staining in next month. The yield of foci and parameters on their morphology intensity and size. Furthermore, each cell can transfer features by cell communication bystander cells. H2ax foci with essentially no change from 10 h to 48 h postirradiation. H2ax foci in irradiated cells, and the results agree well those obtained by manual counts. Bclaf1 is a radiationinduced h2axinteracting partner.
H2ax bands were normalized to the total h2ax bands. Download platform independent to install imagej on a computer with java preinstalled, or to upgrade to the latest full distribution including macros, plugins and luts, download the zip archive 6mb and extract the imagej directory. Image pocessing toolbox for matlab, imagej, mij, java virtual machine. H2ax foci was detected by manually labeling the nucleus using the imagej software. This subreddit is place to discuss image analysis, software features, to get help, and to share ideas, papers, resources. Autofoci, an automated highthroughput foci detection approach for. How to count gamma h2ax foci in cells with the help of. Immunoblot bands were quantitated with nih imagej software. Comet assay and foci dna damage quantification metasystems.
Imagej is an open source image processing program designed for scientific multidimensional images. A standard imagej plugin filter that operates on an opened image. In this study, a characterization of the induction of. We describe an automated fluorescence microscopy system, including automated image processing, to count. There are numerous software solutions for the analysis of foci. This video was inspired by anna mcleans 2011 academic excellence conference presentation. The number of radioinduced doublestrand breaks is correlated with the number of histone gamma h2ax. Rad51 foci formation in response to dna damage is modulated by tip49. They are accompanied by the phosphorylation of the histone 2ax h2ax. H2ax ionizing radiationinduced foci irif following exposure to xrays is presented. Imagej is a free open source application to process images.
A focus detection algorithm with automated parameter. The image processing is performed by a hough transform based algorithm, charm, which has wide applicability for the detection and analysis of cells and cell colonies. H2ax and suppresses dna double strand break repair. Assessing the validity of clinician advice that patients. H2ax and tunelpositive cells in immunofluorescent images was performed using imagej software national institutes of health. We used imagej as independent foci counting program as well as a part of foco. Use the helpupdate imagej command to upgrade to newer versions. Linking gammah2ax foci and cancer in rat skin exposed to. Computational analysis of the number, area and density of. Support is available on the mailing list and on the image. H2ax foci per cell of dapi stained cells in imagej. The dna doublestrand break dsb is the primary lethal lesion after therapeutic radiation. H2ax expands a large distance from the dsb site, and since specific. H2ax foci in breast cancer cells exposed to 111indtpahegf or.
The number of foci per cell was calculated by dividing the total number of foci in the frame to the number of cells containing foci. Ijms free fulltext histone h2ax is involved in foxo3a. Carpenter ae, jones tr, lamprecht mr, clarke c, kang ih, friman o, et al. H2ax, a histone h2a variant, has a key role in the cellular response to dna doublestrand breaks dsbs. Wildtype p53induced phosphatase 1 dephosphorylates histone variant. The quantitative analysis of foci plays an important role in many cell biological methods such as counting of colonies or cells, organelles or vesicles, or the number of protein complexes. Welecome all brothers and sisters from imagej mailing list. The system also relies heavily on robust control software, written using multi.
Reilly 2009 computational analysis of the number, area and density of gamma h2ax foci in breast cancer cells exposed to 111indtpahegf or gammarays using image j software, int. Clsm, images were analyzed with the fiji software schindelin et al. Cellprofiler free opensource software for measuring and. The focinator a new opensource tool for highthroughput foci. Induction and repair of dna doublestrand breaks assessed. However, the first generation assay involves the use of immunofluorescent. Different crosssections of a cell called zstacks were scanned by the lsm system and. How to count gamma h2ax foci in cells with the help of imagej. H2ax, via an antibody, visualises the formation of these foci, allowing the quantification of dna dsbs and forming the basis for a sensitive biological dosimeter of ionising radiation. A quick tutorial on using imagej to count cells automatically.
Image processing and control software for highcontent screening. Tested on gamma h2ax and 53bp1 foci induced by ionization. Finally, the information generated by the above analysis algorithms can be employed to evaluate colocalization between foci formed by different proteins. Gamma h2ax foci in cells exposed to a mixed beam of xrays and alpha particles. All these chemicals reduced viability in a given dose range, but did not induce, even at the highest toxic dose level. Induction and processing of the radiationinduced gamma. Results are presented as a mask image, shown in tabular format andor saved to file. How to count gamma h2ax foci in cells with the help of imagej software. H2ax foci, which can be observed and counted with a fluorescence microscope.
H2ax foci can be used to detect the genotoxic effect of different toxic substances, including ionizing radiation. Wildtype p53induced phosphatase 1 dephosphorylates. Thus, the development of assays to detect and to quantitate these lesions could have broad preclinical and clinical impact. A validation of the software is presented for radiationinduced. This program also can be used for producing foci quantifications for low. H2ax to detect radiationinduced doublestranded dna breaks and with tunel assay to detect apoptotic cells emethods 1 in the supplement. Irif were analyzed by a module written in software imagej, version 1. Cellprofiler is a free opensource software for measuring and analyzing cell images. For this reason, foci quantification is a useful tool to measure radiationinduced dna damage and the number of foci has been suggested as. Surprisingly, unlike human granulocytes, rat granulocytes with dnapkcs deficiency displayed a rapid accumulation, but delayed disappearance of. This subreddit is place to discuss image analysis, software features, to get help, and to share ideas, papers, resources, projects, and expertise. H2ax foci and mni in tk6 cells was established, and the assay provided information on the aneugenicclastogenic mode of action. Itcn imagebased tool for counting nuclei is an imagej plugin for counting the number cells within an image. H2ax foci in three widely used human cell lines and colocalization tested with rad51 and.
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