Details

Autor(en) / Beteiligte

• Pereira, Pedro D
• Serra-Caetano, Ana
• Cabrita, Marisa
• Bekman, Evguenia
• Braga, José
• Rino, José
• Santus, Renè
• Filipe, Paulo L
• Sousa, Ana E
• Ferreira, João A

Titel

Quantification of cell cycle kinetics by EdU (5-ethynyl-2'-deoxyuridine)-coupled-fluorescence-intensity analysis

Ist Teil von

• Oncotarget, 2017-06, Vol.8 (25), p.40514-40532

Ort / Verlag

United States: Impact Journals LLC

Erscheinungsjahr

2017

Quelle

Free E-Journal (出版社公開部分のみ）

Beschreibungen/Notizen

• We propose a novel single-deoxynucleoside-based assay that is easy to perform and provides accurate values for the absolute length (in units of time) of each of the cell cycle stages (G1, S and G2/M). This flow-cytometric assay takes advantage of the excellent stoichiometric properties of azide-fluorochrome detection of DNA substituted with 5-ethynyl-2'-deoxyuridine (EdU). We show that by pulsing cells with EdU for incremental periods of time maximal EdU-coupled fluorescence is reached when pulsing times match the length of S phase. These pulsing times, allowing labelling for a full S phase of a fraction of cells in asynchronous populations, provide accurate values for the absolute length of S phase. We characterized additional, lower intensity signals that allowed quantification of the absolute durations of G1 and G2 phases.Importantly, using this novel assay data on the lengths of G1, S and G2/M phases are obtained in parallel. Therefore, these parameters can be estimated within a time frame that is shorter than a full cell cycle. This method, which we designate as EdU-Coupled Fluorescence Intensity (E-CFI) analysis, was successfully applied to cell types with distinctive cell cycle features and shows excellent agreement with established methodologies for analysis of cell cycle kinetics.