Flow cytometric analysis of the early phases of apoptosis by cellular and nuclear techniques.
Flow cytometric analysis of the early phases of apoptosis by cellular and nuclear techniques.
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DOI：
10.1002/(sici)1097-0320(19960601)24:2
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发表时间：
1996-06
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影响因子：
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通讯作者：
C. Ferlini;S. Cesare;G. Rainaldi;W. Malorni;P. Samoggia;R. Biselli;A. Fattorossi
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文献类型：
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作者：
C. Ferlini;S. Cesare;G. Rainaldi;W. Malorni;P. Samoggia;R. Biselli;A. Fattorossi
在新鲜分离的辐照人淋巴细胞，生长因子剥夺培养的人淋巴细胞和地塞米松（DEX）处理的小鼠胸腺细胞的细胞凋亡过程中发生的早期事件，在质膜，染色质和线粒体水平进行了研究。在完整的，未固定的细胞，光散射特性和DNA的溴化乙锭（EB）的评价允许一个细胞亚群提示初始凋亡被确定。这些细胞的凋亡性质通过在照射的人淋巴细胞模型中的细胞分选来证实。EB不能被碘化丙啶取代，表明所用DNA探针的性质对于检测初始凋亡变化是非常重要的。由于线粒体被认为是细胞凋亡过程中的主要靶点，我们测量了线粒体跨膜电位敏感（罗丹明123）和非敏感（10-壬基-吖啶-橙子）探针的摄取，同时EB摄取。开始凋亡的细胞具有增强的两种线粒体染料的掺入，其与EB组合鉴定了几个细胞亚群。这表明线粒体结构和功能的复杂改变发生在细胞凋亡的早期阶段。为了研究在细胞凋亡的相似阶段中染色质水平上发生的现象，破坏了照射的人淋巴细胞和DEX处理的小鼠胸腺细胞，并在核悬浮液中评估了DNA含量。DNA酶的瞬时增加，即，在细胞凋亡的早期阶段观察到人模型中明显的超二倍体峰的出现和鼠模型中G 0/1峰的普遍上移，伴随着光散射性质的特定改变。这些发现表明，染色质质地改变，在早期细胞凋亡和影响DNA的降解。
The early events occurring during apoptosis at the plasma membrane, chromatin, and mitochondrial levels were investigated in freshly isolated irradiated human lymphocytes, growth factor-deprived cultured human lymphocytes, and dexamethasone (DEX)-treated murine thymocytes. In intact, unfixed cells, evaluation of the light scatter properties and of DNA stainability with ethidium bromide (EB) allowed a cell subset suggestive for initial apoptosis to be identified. The apoptotic nature of these cells was confirmed by cell sorting in irradiated human lymphocyte model. EB could not be substituted for by propidium iodide, indicating that the nature of DNA probe used is of major importance for detecting initial apoptotic changes. Because mitochondria are thought to represent a primary target during apoptosis, we measured the uptake of mitochondria transmembrane potential sensitive (Rhodamine 123) and nonsensitive (10-nonyl-acridine-orange) probes concomitantly with EB uptake. Cells starting apoptosis had an enhanced incorporation of both mitochondria dyes, which in combination with EB identified several cell subsets. This suggests that complex alterations in mitochondrial structure and functioning occur in the early stages of apoptosis. To investigate phenomena occurring at the chromatin level in similar phases of apoptosis, irradiated human lymphocytes and DEX-treated murine thymocytes were disrupted and DNA stainability assessed in nuclear suspensions. A transient increase in DNA stainability, i.e., the appearance of distinct hyperdiploid peaks in the human model and a generalised upward shift of the G0/1 peak in the murine model, was observed in the early phases of apoptosis concomitantly with specific alterations in light scattering properties. These findings suggest that chromatin texture is altered in early apoptosis and affects DNA stainability.