PuSH - Publication Server of Helmholtz Zentrum München: Measurement of the kinetics of DNA repair synthesis after uv irradiation using immunochemical staining of incorporated 5-bromo-2′-deoxyuridine and flow cytometry.

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Beisker, W.* ; Hittelman, W.N.

Measurement of the kinetics of DNA repair synthesis after uv irradiation using immunochemical staining of incorporated 5-bromo-2′-deoxyuridine and flow cytometry.

Exp. Cell Res. 174, 156-167 (1988)

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Open Access Green as soon as Postprint is submitted to ZB.

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The kinetics of unscheduled DNA synthesis in normal human fibroblasts was characterized by flow cytometry utilizing the immunofluorescent detection of 5-bromo-2′-deoxyuridine (BrdUrd) incorporated into cellular DNA during the repair process. Quiescent normal human fibroblasts were irradiated with ultraviolet light and incubated in the presence of BrdUrd during a postirradiation repair period. The amount of unscheduled DNA synthesis was then quantified in the quiescent cells by immunofluorescence staining using monoclonal antibodies against BrdUrd incorporated into the DNA. Significant amounts of unscheduled DNA synthesis were measured after doses as low as 0.1 J/m2 and for time periods as short as 15 min. The initial repair rate was found to be linear with time at all doses tested until repair neared completion. Interestingly, the initial repair rate was constant for doses over the range of 5 to 40 J/m2, whereas the time to completion of repair was dose dependent. These results suggest that above 5 J/m2 in normal human fibroblasts, the repair process is saturated but continues to function until all available regions are repaired. Using this methodology for measuring unscheduled DNA synthesis in combination with second and third flow markers, it is now possible to measure unscheduled DNA synthesis in heterogeneous mixtures of cells.

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Publication type Article: Journal article

Document type Scientific Article

Corresponding Author

ISSN (print) / ISBN 1090-2422

e-ISSN 0014-4827

Journal Experimental Cell Research

Quellenangaben Volume: 174, Issue: 1, Pages: 156-167

Publisher Academic Press

Publishing Place Orlando, Fla.

Non-patent literature Publications

Reviewing status Peer reviewed

Institute(s) Departments & Institutes