Dynamic proliferation assessment in flow cytometry.
Dynamic proliferation assessment via flow cytometry is legitimately supposed to be the most powerful tool for recording cell cycle kinetics in-vitro. The preeminent feature is a single cell-based multi-informative analysis by temporal high-resolution. Flow cytometric approaches are based on labeling of proliferating cells via thymidine substitution by a base analog (e.g., 5-bromo-2'-deoxyuridine, BrdU) that is added to cell cultures either for a short period of time (pulse labeling) or continuously until cell harvesting. This unit describes the alternative use of the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) in place of BrdU for three different applications: (1) dynamic proliferation assessment by EdU pulse cell labeling; (2) the same approach as (1) but in combination with live/dead cell discrimination; and (3) dynamic cell cycle analysis based on continuous cell labeling with EdU and Hoechst fluorochrome quenching. In contrast to the detection of BrdU incorporation, EdU-positive cells can be identified by taking advantage of click chemistry, which facilitates a simplified and fast cell preparation. Further analysis options but also limitations of the utilization of EdU are discussed.
Diermeier-Daucher
Simone
S
Department of Gynaecology and Obstetrics, University of Regensburg, Regensburg, Germany.
Brockhoff
Gero
G
D000818:Animals; D006690:Bisbenzimidazole / Q000032:analysis / Q000737:chemistry; D001973:Bromodeoxyuridine / Q000032:analysis / Q000737:chemistry; D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002477:Cells / Q000166:cytology; D003857:Deoxyuridine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans
D001973:Bromodeoxyuridine; C031086:5-ethynyl-2'-deoxyuridine; D006690:Bisbenzimidazole; D003857:Deoxyuridine