Flow-cytometric assessment of cellular poly(ADP-ribosyl)ation capacity in peripheral blood lymphocytes
- Equal contributors
1 Molecular Toxicology Group, Department of Biology, Box X911, University of Konstanz, D-78457 Konstanz, Germany
2 Cancer and Ageing Research Group, School of Biomedical Sciences, University of Ulster, Coleraine, BT52 1SA, Northern Ireland, UK
3 School of Biomedical and Natural Sciences, College of Science and Technology, Nottingham Trent University, Clifton Campus, NG11 8NS, Nottingham, UK
Immunity & Ageing 2006, 3:8 doi:10.1186/1742-4933-3-8Published: 19 July 2006
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.
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.
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.