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.
© 2011 by John Wiley & Sons, Inc.