Chrest F J, Buchholz M A, Kim Y H, Kwon T K, Nordin A A
Clinical Immunology Section, National Institute on Aging, NIH, Baltimore, Maryland 21224, USA.
Cytometry. 1995 May 1;20(1):33-42. doi: 10.1002/cyto.990200107.
Light scatter measurements using flow cytometry indicated that T cells from young and old mice undergo apoptosis following activation with immobilized anti-CD3. The percentage of cells in apoptosis after 20 h activation was significantly greater (p < .001) in cultures containing cells from older animals. The mean percentages of apoptotic T cells from young and old mice after 20 h activation were 19.3% and 33.0%, respectively. The proportion of viable cells after 20 h activation was significantly higher (p < .003) in the young (mean = 78.4%) than in the old animals (mean = 65.8%). Simultaneous measurements of light scatter and fluorescence indicated that apoptotic T cells contained both the CD4+ and the CD8+ T-cell phenotypes. The frequency of apoptotic CD8+ T cells was elevated (p < .007) in older animals, where the mean percentage was 15.1%, compared to 5.3% in the young. The most dramatic difference between young and old (p < .0008) was seen in the percentages of viable CD4+ T cells after 20 h activation. The mean viable CD4+ T-cell percentage was 33.7% in- the young and 21.4% in the old. CD4+ cells expressing high levels of CD45RB (CD45RBhi) after activation for 20 h possessed light scatter and bright fluorescence properties characteristic of viable cells, whereas CD4+/CD45RBlo density cells could be identified as apoptotic based on their decreased CD4 fluorescence and scatter characteristics. CD4+ cells from young animals were predominantly CD45RBhi, whereas CD4+ cells from the old had greater levels of CD454RBlo cells. In addition to light scatter changes, measurement of DNA content after 40 h activation revealed the presence of a sub-G1 DNA apoptotic peak and a viable cell cycle distribution. After 40 h of activation, there was an increase in the percentage of apoptotic cells in both young and old mice, with the greatest increase seen in the cells from older animals. Further evidence supporting the process of apoptosis in 40 h-activated cells was confirmed by the appearance of DNA strand breaks detected by in situ nick translation.
使用流式细胞术进行的光散射测量表明，来自年轻和老年小鼠的T细胞在用固定化抗CD3激活后会发生凋亡。在含有老年动物细胞的培养物中，激活20小时后凋亡细胞的百分比显著更高（p <.001）。激活20小时后，年轻和老年小鼠凋亡T细胞的平均百分比分别为19.3%和33.0%。激活20小时后，年轻动物（平均值 = 78.4%）中活细胞的比例显著高于老年动物（平均值 = 65.8%）（p <.003）。光散射和荧光的同步测量表明，凋亡T细胞同时包含CD4+和CD8+ T细胞表型。老年动物中凋亡CD8+ T细胞的频率升高（p <.007），其平均百分比为15.1%，而年轻动物中为5.3%。年轻和老年之间最显著的差异（p <.0008）出现在激活20小时后活CD4+ T细胞的百分比上。年轻动物中活CD4+ T细胞的平均百分比为33.7%，老年动物中为21.4%。激活20小时后表达高水平CD45RB（CD45RBhi）的CD4+细胞具有活细胞特有的光散射和明亮荧光特性，而CD4+/CD45RBlo密度细胞可根据其降低的CD4荧光和散射特性被鉴定为凋亡细胞。来自年轻动物的CD4+细胞主要是CD45RBhi，而来自老年动物的CD4+细胞中CD454RBlo细胞的水平更高。除了光散射变化外，激活40小时后DNA含量的测量揭示了亚G1期DNA凋亡峰的存在和活细胞周期分布。激活40小时后，年轻和老年小鼠中凋亡细胞的百分比均增加，老年动物细胞中的增加最为明显。通过原位缺口平移检测到的DNA链断裂的出现证实了支持40小时激活细胞中凋亡过程的进一步证据。
