Cluster Size Analysis for γH2AX Assay: A Novel Tool for DNA Damage Quantification in Cells with Diverse Radiosensitivity

Cluster Size Analysis for γH2AX Assay: A Novel Tool for DNA Damage Quantification in Cells with Diverse Radiosensitivity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Cluster Size Analysis for γH2AX Assay: A Novel Tool for DNA Damage Quantification in Cells with Diverse Radiosensitivity Joanna Szczechowska, Beata Brzozowska, Małgorzata Kopeć, Beata Pszczółkowska-Kępa, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7434384/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Introduction : DNA double-strand breaks (DSBs) are considered the most lethal of all DNA lesions and are responsible for the bulk of cytotoxic effects induced by ionizing radiation (IR) and cancer chemotherapeutics. Objectives: This study focuses on radiation-induced DNA damage in glioblastoma cell lines characterized by disparate radiosensitivity, where divergent degrees of complex damage induction were observed. Methods: Fluorescence microscopy evaluation showed an expected linear increase in the relative foci number, localization, and signal intensities in cells with absorbed doses up to 6 Gy, albeit with repeatable pattern variation among the different cell lines. The cluster size parameter was introduced as a novel quantification approach for γH2AX assay analysis when considerable discrepancies in cell sensitivities are persistent. Results: This approach provides a robust tool to differentiate dose-response shape results, allowing for the distinction of seemingly similar responses. Conclusion: The method, addressing each cell line's unique cluster size distribution, is thus recommended for granular analysis of γH2AX foci in multiple cell lines of similar origin, demonstrating its impact on therapeutic responses in heterogeneous tumors. Graphical Abstract: glioblastoma multiforme (GBM) cancer cell radiosensitivity DNA double-strand breaks (DSBs) γH2AX assay X-ray radiation effects cluster size analysis Full Text Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7434384","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":511809486,"identity":"d7e10cef-ca06-416c-a145-acee4714128e","order_by":0,"name":"Joanna Szczechowska","email":"","orcid":"","institution":"The Maria Sklodowska-Curie National Research Institute of Oncology","correspondingAuthor":false,"prefix":"","firstName":"Joanna","middleName":"","lastName":"Szczechowska","suffix":""},{"id":511809487,"identity":"4d127c27-a6db-44a5-8d3e-a449838dcab9","order_by":1,"name":"Beata Brzozowska","email":"","orcid":"","institution":"University 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γH2AX assay, X-ray radiation effects, cluster size analysis","lastPublishedDoi":"10.21203/rs.3.rs-7434384/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7434384/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eIntroduction\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eDNA double-strand breaks (DSBs) are considered the most lethal of all DNA lesions and are responsible for the bulk of cytotoxic effects induced by ionizing radiation (IR) and cancer chemotherapeutics.\u003c/p\u003e\u003cp\u003eObjectives:\u003c/p\u003e\u003cp\u003eThis study focuses on radiation-induced DNA damage in glioblastoma cell lines characterized by disparate radiosensitivity, where divergent degrees of complex damage induction were observed.\u003c/p\u003e\u003cp\u003eMethods:\u003c/p\u003e\u003cp\u003eFluorescence microscopy evaluation showed an expected linear increase in the relative foci number, localization, and signal intensities in cells with absorbed doses up to 6 Gy, albeit with repeatable pattern variation among the different cell lines. The cluster size parameter was introduced as a novel quantification approach for γH2AX assay analysis when considerable discrepancies in cell sensitivities are persistent.\u003c/p\u003e\u003cp\u003eResults:\u003c/p\u003e\u003cp\u003eThis approach provides a robust tool to differentiate dose-response shape results, allowing for the distinction of seemingly similar responses. 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