A bromodeoxyuridine (BUdR)-mithramycin technique for detecting cycling and non-cycling cells by flow microfluorometry

Bromodeoxyuridine (BUdR) incorporation into cellular DNA results in an increase in fluorescence of mithramycin-stained cells. Flow microfluorometry can be used to distinguish among cells which have not replicated, cells which have replicated once, and cells which have replicated two or more times in the presence of 30 μM BUdR. This technique offers two advantages over present protocols: (1) a positive fluorescent signal is provided by the mithramycin stain rather than a negative signal, as with the Hoechst stain; (2) after mithramycin staining, hundreds of thousands of cells in any phase of the cell cycle can be analyzed in less than 10 min. The BUdR-mithramycin technique described herein is a useful tool for studying the cell-cycle kinetics of heterogeneous populations containing dividing and non-dividing cells.