Cell cycle analysis by combining the 5-bromodeoxyuridine/33258 Hoechst technique with DNA-specific ethidium bromide staining

Cells were grown in 5-bromodeoxyuridine(BrdUrd)-containing medium, harvested and stained with a combination of 33258 Hoechst and ethidium bromide for analysis in a two-parametrical flow cytometer. Both fluorescences were excited by UV laser light, and ethidium bromide was additionally excited by energy transfer from the Hoechst dye. The separation of the two fluorescences proved to be excellent, the projected histograms showing a quality comparable to the quality obtained by single dye staining. The ethidium bromide fluorescence gives information as to where the cell is located within the cycle of DNA replication, while the BrdUrd-quenched Hoechst fluorescence gives information as to where in the cycle the cell was located at the beginning of BrdUrd incorporation. In this way information is obtained concerning the distance a cell traveled through the cycle during the BrdUrd incubation time. This method may become a powerful tool in many investigations dealing with cell cycle perturbations in culture.