Flow cytometric ratio analysis of the Hoechst 33342 emission spectrum: Multiparametric characterization of apoptotic lymphocytes

The apoptotic response to various stimuli is an important part of immune regulation, and the ability to identify apoptotic lymphocytes within a complex population is a prerequisite to a more detailed understanding of its role in vivo. We describe a flow cytometric technique which utilizes viable cells and enables simultaneous identification of apoptotic cells and analyses of immunophenotype, cell cycle progression, membrane integrity and light scatter properties. It is based upon analysis of two regions of the emission spectrum of the DNA-binding vital dye Hoechst 33342. We established a precise correlation between the ratio of red to blue fluorescence emission and apoptosis based upon nuclear morphology and the presence of characteristic DNA degradation patterns. In human peripheral blood lymphocytes (PBL) and mouse thymocytes we incorporated light scatter properties, cell cycle stage, relevant cell surface immunophenotypic markers (CD25 or CD4 and CD8) and a marker of plasma membrane integrity (merocyanine 540) to enable multiparametric phenotyping of apoptotic cells, We show that staurosporine-induced apoptosis of ConA-stimulated PBL is not correlated with cell cycle stage but is selective for activated cells since the frequency of large, CD25(+) cells is decreased by staurosporine. Dexamethasone and ionomycin differ in their ability to induce apoptosis selectively in murine thymocyte subsets. Dexamethasone kills a broad spectrum of the CD4/8 immunophenotypes with no selectivity for cell cycle stage. Ionomycin selectively depletes CD4(+)8(+) cells, especially those in the G(0)/G(1) region of the cell cycle, and spared CD4(-)8(+) cells. This technique is broadly advantageous for in vitro and ex vivo models of apoptosis in that it interrogates individual viable cells and correlates membrane and nuclear apoptotic changes with standard flow cytometric immunophenotyping.