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Flow-cytometric assessment of cellular poly(ADP-ribosyl)ation capacity in peripheral blood lymphocytes

Flow-cytometric assessment of cellular poly(ADP-ribosyl)ation capacity in peripheral blood lymphocytes

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KUNZMANN, Andrea, Dan LIU, Kathryn ANNETT, Muriel MALAISÉ, Bastian THAA, Paul HYLAND, Yvonne BARNETT, Alexander BÜRKLE, 2006. Flow-cytometric assessment of cellular poly(ADP-ribosyl)ation capacity in peripheral blood lymphocytes. In: Immunity & Ageing. 3(1), 8. ISSN 1742-4933. eISSN 1742-4933

@article{Kunzmann2006Flow--28144, title={Flow-cytometric assessment of cellular poly(ADP-ribosyl)ation capacity in peripheral blood lymphocytes}, year={2006}, doi={10.1186/1742-4933-3-8}, number={1}, volume={3}, issn={1742-4933}, journal={Immunity & Ageing}, author={Kunzmann, Andrea and Liu, Dan and Annett, Kathryn and Malaisé, Muriel and Thaa, Bastian and Hyland, Paul and Barnett, Yvonne and Bürkle, Alexander}, note={Article Number: 8} }

Annett, Kathryn Thaa, Bastian Liu, Dan 2014-06-26T08:10:54Z 2014-06-26T08:10:54Z Barnett, Yvonne eng Bürkle, Alexander Malaisé, Muriel Malaisé, Muriel Thaa, Bastian Kunzmann, Andrea Annett, Kathryn Immunity & Ageing ; 3 (2006). - 8 Barnett, Yvonne 2006 Kunzmann, Andrea Hyland, Paul Hyland, Paul Flow-cytometric assessment of cellular poly(ADP-ribosyl)ation capacity in peripheral blood lymphocytes Background<br /><br /><br />Poly(ADP-ribosyl)ation is a posttranslational modification of nuclear proteins catalysed by poly(ADP-ribose) polymerases (PARPs), using NAD+ as a substrate. Activation of PARP-1 is in immediate response to DNA damage generated by endogenous and exogenous damaging agents. It has been implicated in several crucial cellular processes including DNA repair and maintenance of genomic stability, which are both intimately linked with the ageing process. The measurement of cellular poly(ADP-ribosyl)ation capacity, defined as the amount of poly(ADP-ribose) produced under maximal stimulation, is therefore relevant for research on ageing, as well as for a variety of other scientific questions.<br /><br /><br /><br />Results<br /><br /><br />This paper reports a new, robust protocol for the measurement of cellular poly(ADP-ribosyl)ation capacity in PBMC or Jurkat T-cells using flow cytometry, based on a previously established immuno-dot-blot assay. In order to validate the new assay, we determined the dose-response curve of 3-aminobenzamide, a well-known competitive PARP inhibitor, and we derived an IC50 that is very close to the published value. When testing a set of PBMC samples taken from fifteen healthy young human donors, we could confirm the presence of a substantial interindividual variation, as previously observed using a radiometric assay.<br /><br /><br /><br />Conclusion<br /><br /><br />The methodology described in this paper should be generally useful for the determination of cellular poly(ADP-ribosyl)ation capacity in a wide variety of settings, especially for the comparison of large sets of samples, such as population studies. In contrast to previously published radiometric or immuno-dot-blot assays, the new FACS-based method allows (i) selective analysis of mononuclear cells by gating and (ii) detection of a possible heterogeneity in poly(ADP-ribosyl)ation capacity between cells of the same type. Bürkle, Alexander deposit-license Liu, Dan

Dateiabrufe seit 01.10.2014 (Informationen über die Zugriffsstatistik)

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