Cell type specific applicability of 5-ethynyl-2'-deoxyuridine (EdU) for dynamic proliferation assessment in flow cytometry.
Using the nucleoside analogue EdU (5-ethynyl-2'-deoxyuridine) for thymidine substitution instead of BrdU (5-bromo-2'-deoxyuridine) in cell proliferation assays has recently been proposed. However, the effect of EdU on cell viability, DNA synthesis, and cell cycle progression and consequently its usability for dynamic cell proliferation analysis in vitro has not been explored. We compared the effect of EdU and BrdU incorporation into SK-BR-3 and BT474 breast cancer cells and the impact on cell cycle kinetics, cell viability, and DNA damage. We found that EdU can be used not only for pulse but also for continuous cell labeling and henceforth in high resolution EdU/Hoechst quenching assays. BrdU and EdU proliferation assays based on click chemistry revealed comparable results. However, cell viability of SK-BR-3 breast cancer cells was highly affected by long term exposure to EdU. Both SK-BR-3 as well as BT474 cells show cell cycle arrests upon long term EdU treatment whereas only SK-BR-3 cells were driven into necrotic cell death by long term exposure to EdU. In contrast BT474 cells appeared essentially unharmed by EdU treatment in terms of viability. Consequently using EdU enables highly sensitive and quantitative detection of proliferating cells and facilitates even continuous cell cycle assessment. Nevertheless, potential cellular susceptibility needs to be individually evaluated.
Diermeier-Daucher
Simone
S
Department of Obstetrics and Gynecology, University of Regensburg, Germany.
Clarke
Scott T
ST
Hill
Dani
D
Vollmann-Zwerenz
Arabel
A
Bradford
Jolene A
JA
Brockhoff
Gero
G
D017209:Apoptosis; D001973:Bromodeoxyuridine / Q000633:toxicity; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D019926:Cyclin B / Q000187:drug effects / Q000378:metabolism; D056744:Cyclin B1; D004247:DNA / Q000378:metabolism; D003857:Deoxyuridine / Q000031:analogs & derivatives* / Q000633:toxicity; D005260:Female; D005434:Flow Cytometry; D006657:Histones / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology
C534699:CCNB1 protein, human; D019926:Cyclin B; D056744:Cyclin B1; C468783:H2AX protein, human; D006657:Histones; D004247:DNA; D001973:Bromodeoxyuridine; C031086:5-ethynyl-2'-deoxyuridine; D003857:Deoxyuridine