title: Dual-pulse labeling using 5-ethynyl-2'-deoxyuridine (EdU) and 5-bromo-2'-deoxyuridine (BrdU) in flow cytometry.
abstract: Changes in DNA replication during S-phase can give insights into mechanisms of cell growth, cell cycle kinetics, and cytotoxicity. A common method for detection of cell proliferation utilizes the incorporation of a thymidine analog during DNA synthesis. Incorporation of multiple analogs at different time points can further define cell cycle kinetics. Traditionally, the dual-pulse method has been done by combining 5-bromo-2'-deoxyuridine (BrdU) with iododeoxyuridine or chlorodeoxyuridine, with detection using multiple cross-reacting BrdU antibodies. This unit presents a dual-pulse method using the thymidine analog 5-ethyl-2'-deoxyuridine (EdU), detected by click chemistry, combined with BrdU labeling and detection. No cross reactivity with incorporated EdU is observed using the BrdU antibody clone MoBU-1. EdU detection using click chemistry does not cross-react with incorporated BrdU. Cells are first pulsed with EdU, and then pulsed with BrdU; sequential pulses of EdU, followed by BrdU, are done without removing or washing out EdU.
authors.affiliation: Life Technologies, Eugene, Oregon.
authors.forename: Jolene A
authors.initials: JA
authors.lastname: Bradford
authors.affiliation: Life Technologies, Eugene, Oregon.
authors.forename: Scott T
authors.initials: ST
authors.lastname: Clarke
mesh_terms: D001973:Bromodeoxyuridine / Q000737:chemistry; D049109:Cell Proliferation; D002477:Cells / Q000166:cytology; D057930:Click Chemistry / Q000379:methods*; D003857:Deoxyuridine / Q000031:analogs & derivatives* / Q000737:chemistry; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008722:Methods; D015336:Molecular Probe Techniques; D016196:S Phase; D013194:Staining and Labeling / Q000379:methods
chemical_list: D001973:Bromodeoxyuridine; C031086:5-ethynyl-2'-deoxyuridine; D003857:Deoxyuridine