{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,31]],"date-time":"2025-10-31T13:50:25Z","timestamp":1761918625190},"reference-count":32,"publisher":"Wiley","issue":"1","license":[{"start":{"date-parts":[[2004,9,28]],"date-time":"2004-09-28T00:00:00Z","timestamp":1096329600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cytometry Pt A"],"published-print":{"date-parts":[[2004,11]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:sec><jats:title>Background<\/jats:title><jats:p>The induction of DNA double\u2010strand breaks (DSBs) in chromatin triggers histone H2AX phosphorylation (on <jats:italic>Ser<\/jats:italic>\u2010139) by ATM\u2010, ATR\u2010, or DNA\u2010dependent protein kinases (DNA\u2010PK). Phosphorylated H2AX, denoted as \u03b3H2AX, can be detected immunocytochemically using an antibody that is specific to the <jats:italic>Ser<\/jats:italic>\u2010139\u2013phosphorylated epitope. We previously reported that the induction DSBs by DNA topoisomerase I or II inhibitors can be monitored in individual cells by measuring \u03b3H2AX immunofluorescence (IF) by cytometry. The present study explored whether the detection of \u03b3H2AX IF can serve as a marker of the presence of the DNA precursor bromodeoxyuridine (BrdU) that is incorporated into DNA.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>HeLa cells growing on microscope slides were incubated with BrdU for 1 h, rinsed free of the precursor, and incubated for different periods for up to 12 h. The cells were then briefly incubated with Hoechst 33342 (to sensitize BrdU\u2010labeled DNA to ultraviolet [UV] light), irradiated with 300 nm UV light to photolyze BrdU\u2010labeled DNA, transferred back into culture for an additional hour, and fixed. Cells were concurrently immunostained for \u03b3H2AX (Alexa Fluor 633) and cyclin A (fluorescein isothiocyanate); their DNA was counterstained with 4,6\u2010diamidino\u20102\u2010phenylindole. The intensities of cellular far red (\u03b3H2AX), green (cyclin A), and blue (DNA) fluorescences were measured by laser scanning cytometry.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>After a 1\u2010h pulse of BrdU followed by exposure to UV, nearly all cells with S\u2010phase DNA content had many\u2010fold higher \u03b3H2AX IF than G<jats:sub>1<\/jats:sub> or G<jats:sub>2<\/jats:sub>\/M cells. The nonirradiated cells had minimal (\u201cprogrammed\u201d) expression of \u03b3H2AX, whereas the irradiated cells incubated without BrdU had uniformly elevated levels of \u03b3H2AX IF independent of the cell cycle phase. Pulse\u2010chase experiments showed that the cohort of BrdU\u2010labeled (\u03b3H2AX\u2010positive) cells progressed through G<jats:sub>2<\/jats:sub>\/M and into G<jats:sub>1<\/jats:sub> phase after 8 and 12 h of growth in BrdU\u2010free medium, respectively. Bivariate analysis of \u03b3H2AX versus cyclin A expression for the gated S\u2010phase cells showed a correlation between these variables, suggesting that the rate of BrdU incorporation (DNA replication) correlates with expression of cyclin A.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions<\/jats:title><jats:p>Photolysis of BrdU\u2010labeled DNA induces DSBs and leads to H2AX phosphorylation. Detection of \u03b3H2AX IF indicates the presence of the incorporated BrdU, is compatible with concurrent detection of other intracellular antigens, and can be used to demonstrate cell cycle kinetics. \u00a9 2004 Wiley\u2010Liss, Inc.<\/jats:p><\/jats:sec>","DOI":"10.1002\/cyto.a.20086","type":"journal-article","created":{"date-parts":[[2004,9,28]],"date-time":"2004-09-28T19:02:36Z","timestamp":1096398156000},"page":"1-7","source":"Crossref","is-referenced-by-count":17,"title":["Histone H2AX phosphorylation induced by selective photolysis of BrdU\u2010labeled DNA with UV light: Relation to cell cycle phase"],"prefix":"10.1002","volume":"62A","author":[{"given":"Xuan","family":"Huang","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Malcolm","family":"A. King","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"H. 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