A flow cytometric method using Hoechst 33342 and propidium iodide for simultaneous cell cycle analysis and apoptosis determination in unfixed cells

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Abstract
A flow cytometric method to detect apoptotic cells is described. This method is based on the detection of differences in chromatin condensation with Hoechst 33342 as a probe and the detection of dead cells with propidium iodide as a probe for membrane damage. By this method it was possible to detect, in the same sample and at the same time, intact cells, cells undergoing apoptosis, and dead cells resulting from apoptotic and/or necrotic processes. The method was successfully applied to the detection of apoptotic cells in two human cell models: cultured polymorphonuclear cells and the U937 cell line treated with antitumoral drugs. Staining specificity for apoptotic cells was controlled by cell sorting of the presumed apoptotic population, followed by morphologic examination or DNA analysis of the sorted populations. The usefulness of such a method is discussed in terms of applications in the analysis of heterogeneous clinical samples, populations with low DNA degradation during apoptosis, and cell cycle position of the apoptotic cells.