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COMPARISON OF CELLULAR AND NUCLEAR FLOW CYTOMETRIC TECHNIQUES FOR DISCRIMINATING APOPTOTIC SUBPOPULATIONS
Citation:
Elstein, K. AND R. Zucker. COMPARISON OF CELLULAR AND NUCLEAR FLOW CYTOMETRIC TECHNIQUES FOR DISCRIMINATING APOPTOTIC SUBPOPULATIONS. U.S. Environmental Protection Agency, Washington, D.C., EPA/600/J-95/048, 1994.
Description:
We compared cellular flow cytometric methods employing carboxyfluorescein (CF), Hoechst 33342, and Hoechst 33258 with a nuclear method in their ability to discriminate apoptotic subpopulations in rat thymocyte cultures exposed to dexamethasone r tributyltin. n the nuclear technique, apoptotic cells appeared at a single population containing reduced DNA content. while in the cellular techniques, apoptotic cells appeared as two or more subpopulations exhibiting increased fluorescence. f these subpopulations, early apoptotic cells (which excluded propidium excluded propidium iodide [PI] , indicating maintenance of membrane integrity) exhibited higher fluorescence but the same level of axial light loss (i.s. , size) as viable cells; late apoptotic and dead cells (which incorporated PI) exhibited decreased axial light loss. owever, while the Hoechst dyes allowed discrimination of late apoptotic from dead cells, CF did not. n comparing sensitivity to staining conditions, Hoechst 33258 luorescence was tho most stable over time, Hoechst 33342 the least, and CF fluorescence not only varied with time, but with TBT concentration. omparison of single-parameter analyses revealed that axial light loss was sensitive only to late apoptotic changes; nuclear fluorescence was a better indicator of apoptotic subpopulations, but still underestimated the total percentage of affected cells; and Hoechst 33342 distinguished early apoptotic cells as those with elevated fluorescence. arly apoptotic cells stained with Hoechst 33258 also exhibited increased fluorescence, but could not be distinguished from late apoptotic and dead cells without a second parameter. hose findings indicate that of the methods investigated, the method of choice for detecting apoptosis depends on the goal of analysis: Hoechst 33258 was best for discriminating apoptotic subpopulations, and CF for assessing alterations of membrane fluidity. or single-parameter analyses, Hoechst 33258 was best for determining the total percentage of affected cells, while Hoechst 33342 was able to determine the percentage in early apoptosis.