Differential staining of apoptotic nuclei in living cells: Application to macronuclear elimination in Tetrahymena

Acridine orange (AO) has been used as a vital fluorescent stain to identify apoptotic cells in Drosophila, but little is known about what structures are stained. We explored the specificity of AO staining while studying nuclear apoptosis in Tetrahymena. Using AO alone or together with the vital nuclear stain Hoechst 33342 (HO), we find that lysosomes are generally clustered around the degenerating nucleus and that such nuclei are stained an orange-red color, like lysosomes. Significantly, the combined dyes, more so than with AO alone, distinguish between apoptotic and normal (or necrotic) nuclei by a clear color difference. Moreover, these dyes differentially stain apoptotic and normal nuclei in avian chondrocytes. The differential staining results are nullified in fixed cells or in cytoskeletal preparations treated with RNAse. Similarly, lysosomotrophic agents eliminate the differential staining. Our results are consistent with acidification of the apoptotic nucleus, possibly by fusion with lysosomes. However, even under basic conditions, the macronucleus condenses and is eliminated, suggesting that, if the nucleus is becoming acidified, acidification by itself is not essential for nuclear elimination. The differential staining procedure may provide a useful method for specifically identifying apoptotic cells and separating them for further analysis.