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Cytometry A. 2018 Feb;93(2):239-248. doi: 10.1002/cyto.a.23295. Epub 2017 Dec 8.

Multiparameter cytometric analysis of complex cellular response.

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Department of Cytokinetics, Institute of Biophysics of the Czech Academy of Sciences, Brno, Czech Republic.
Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic.
Center of Biomolecular and Cellular Engineering, International Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czech Republic.


Complex analysis of cellular responses after experimental treatment is important for screening, mechanistic understanding of treatment effects, and the identification of sensitive and resistant cell phenotypes. Modern multicolor flow cytometry has demonstrated its power for such analyses. Here, we introduce a multiparametric protocol for complex analysis of cytokinetics by the simultaneous detection of seven fluorescence parameters. This analysis includes the detection of two surface markers for immunophenotyping, analysis of proliferation based on the cell cycle and the measurement of incorporated nucleoside analogue 5-ethynyl-2'-deoxyuridine (EdU) in newly synthesized DNA, analysis of DNA damage using an anti-phospho-histone H2A.X (Ser139) antibody, and determination of cell death using a fixable viability probe and intracellular detection of caspase-3 activation. To demonstrate the applicability of this protocol for the analysis of heterogeneous and complex cell responses, we used different treatments and model cell lines. We demonstrated that this protocol has the potential to provide complex and simultaneous analysis of cytokinetics and analyze the heterogeneity of the response at the single-cell level.


DNA damage; apoptosis; flow cytometry; immunophenotyping; multiparametric analysis; proliferation

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