Measurement of cell-cycle phase-specific cell death using Hoechst 33342 and propidium iodide: preservation by ethanol fixation

We developed a rapid technique for preservation of Hoechst 33342/propidium iodide-stained cells, using ethanol as a fixative. Combined staining with these dyes makes possible analysis of cell-cycle phase-specific cell death. The technique relies on exclusion of propidium iodide from the viable cells, whereas Hoechst stains all of the cells. The bivariate histograms resulting from the flow cytometric analysis contain the equivalent of two single-parameter DNA histograms, one of the living and the other of the dead cell population. Preservation of staining involved addition of 25% ethanol in PBS after propidium iodide staining and before Hoechst staining. The separation between the living and the dead cell populations was maintained for over 3 days at 4 degrees C. This technique will be valuable for quantitative evaluation of the cell-cycle phase-specific effects of cytostatic or cytotoxic agents, particularly in situations where a lag period between staining and analysis is unavoidable.