Combination of BUdR-quenched Hoechst fluorescence with DNA-specific ethidium bromide fluorescence for cell cycle analysis with a two-parameter flow cytometer.
Stimulated or asynchronous L-cells were grown in a BUdR-medium, harvested and stained with a combination of 33258 Hoechst and ethidium bromide for analysis in a FACS II cell sorter. The u.v. laser line served as a light source for exciting the Hoechst fluorescence, the ethidium bromide fluorescence being excited mainly by energy transfer from the Hoechst dye. The quenched Hoechst fluorescence was analysed between 410 nm and 480 nm, the DNA specific EB fluorescence at beyond 630 nm. Thus, not only the actual location of each cell in the cycle could be determined, but also its initial location at time 0 of the experiment, together with its moment of division (BUdR-quenched Hoechst fluorescence). This method could become a powerful tool in many investigations dealing with cell cycle perturbations in culture.