Differential staining of apoptotic nuclei in living cells: Application to macronuclear elimination in Tetrahymena
Differential staining of apoptotic nuclei in living cells: Application to macronuclear elimination in Tetrahymena
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DOI：
10.1177/002215549704500505
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发表时间：
1997-05-01
影响因子：
3.2
通讯作者：
Wolfe, J
中科院分区：
文献类型：
--
作者：
Mpoke, SS;Wolfe, J
吖啶橙（AO）已被用作鉴定果蝇凋亡细胞的重要荧光染色剂，但对染色的结构知之甚少。我们在研究四膜虫细胞核凋亡的同时，探讨AO染色的特异性。单独使用AO或与活体核染色Hoechst 33342 （HO）一起使用，我们发现溶酶体通常聚集在变性核周围，并且这些核被染成橙红色，与溶酶体一样。值得注意的是，与单独使用AO相比，联合染料更能通过明显的颜色差异区分凋亡细胞核和正常（或坏死）细胞核。此外，这些染料对鸟类软骨细胞的凋亡细胞核和正常细胞核的染色存在差异。在固定细胞或用RNAse处理的细胞骨架制剂中，差异染色结果无效。同样，溶酶体营养剂消除差异染色。我们的结果与凋亡细胞核的酸化一致，可能是通过与溶酶体融合。然而，即使在基本条件下，大核也会凝结并被消除，这表明，如果核正在酸化，酸化本身对于核消除并不必要。鉴别染色方法可为特异性鉴定凋亡细胞并将其分离作进一步分析提供一种有用的方法。
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.