Cell Cycle Resolved Measurements of Poly(ADP-Ribose) Formation and DNA Damage Signaling by Quantitative Image-Based Cytometry
Formation of poly(ADP-ribose) (PAR) marks intracellular stress signaling and is notably induced upon DNA damage. PAR polymerases (PARPs) catalyze PAR synthesis upon genotoxic stress and thereby recruit multiple proteins to damaged chromatin. PAR induction is transient and antagonized by the action of PAR glycohydrolase (PARG). Given that poly(ADP-ribosyl)ation (PARylation) is involved in genome integrity maintenance and other vital cellular functions, but also in light of the recent approval of PARP inhibitors for cancer treatments, reliable measurements of intracellular PAR formation have gained importance. Here we provide a detailed protocol for PAR measurements by quantitative image-based cytometry. This technique combines the high spatial resolution of single-cell microscopy with the advantages of cell population measurements through automated high-content imaging. Such upscaling of immunofluorescence-based PAR detection not only increases the robustness of the measurements through averaging across large cell populations but also allows for the discrimination of subpopulations and thus enables multivariate measurements of PAR levels and DNA damage signaling. We illustrate how this technique can be used to assess the dynamics of the cellular response to oxidative damage as well as to PARP inhibitor-induced genotoxicity in a cell cycle resolved manner. Due to the possibility to use any automated microscope for quantitative image-based cytometry, the presented method has widespread applicability in the area of PARP biology and beyond.
Key wordsPoly(ADP-ribose) (PAR) PARP1 ARTD1 PARP inhibitors Olaparib DNA damage Cell cycle Cytometry High-content microscopy Quantitative single-cell analyses
We are grateful to our lab members Thomas Schmid, Federico Teloni, and Stefania Pellegrino for their help with quantitative image-based cytometry and to Jiri Lukas and Luis Toledo for sharing reagents and exchange of unpublished results. Luis Toledo also provided valuable comments on the manuscript. Urs Ziegler and José María Mateos Melero from the Center for Microscopy and Image Analysis at the University of Zurich are acknowledged for expert microscopy support. Research in the lab of Matthias Altmeyer is financed by the Swiss National Science Foundation (SNSF Professorship Grant PP00P3_150690) and by the University of Zurich Association Research Talent Development Fund.
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