COMPARISON OF CELLULAR AND NUCLEAR-FLOW CYTOMETRIC TECHNIQUES FOR DISCRIMINATING APOPTOTIC SUBPOPULATIONS

We compared cellular flow cytometric methods employing carboxyfluoresc ein (CF), Hoechst 33342, and Hoechst 33258 with a nuclear method in th eir ability to discriminate apoptotic subpopulations in rat thymocyte cultures exposed to dexamethasone or tributyltin. In the nuclear techn ique, apoptotic cells appeared as a single population containing reduc ed DNA content, while in the cellular techniques, apoptotic cells appe ared as two or more subpopulations exhibiting increased fluorescence. Of these subpopulations, early apoptotic cells [which excluded propidi um iodide (PI), indicating maintenance of membrane integrity] exhibite d higher fluorescence but the same level of axial light loss (i.e., si ze/refractive index) as viable cells; late apoptotic and dead cells (w hich incorporated PI) exhibited decreased axial light loss. However, w hile the Hoechst dyes allowed discrimination of late apoptotic from de ad cells, CF did not. In comparing sensitivity to staining conditions, Hoechst 33258 fluorescence was the most stable over time, Hoechst 333 42 the least, and CF fluorescence not only varied with time, but with tri-n-butyltin methoxide concentration. Comparison of single-parameter analyses revealed that axial light loss was sensitive only to late ap optotic changes; nuclear fluorescence was a better indicator of apopto tic subpopulations, but still underestimated the total percentage of a ffected cells, and Hoechst 33342 distinguished early apoptotic cells a s those with elevated fluorescence. Early apoptotic cells stained with Hoechst 33258 also exhibited increased fluorescence, but could not be distinguished from late apoptotic and dead cells without a second par ameter. These findings indicate that of the methods investigated, the method of choice for detecting apoptosis depends on the goal of analys is: Hoechst 33258 was best for discriminating apoptotic subpopulations , and CP was best for assessing alterations of membrane fluidity. For single-parameter analyses, Hoechst 33258 was best for determining the total percentage of affected cells, while Hoechst 33342 could be used to determine the percentage in early apoptosis. (C) 1994 Academic Pres s, Inc.