流式细胞术的亚G_1峰法分析细胞凋亡的重新评价

目的重新评价亚G1峰 (Sub G1)在细胞凋亡研究中的应用 ,解决应用中存在的问题。方法采用Sub G1法荧光参数线性放大和对数放大法对 3种具有不同细胞周期特异性的细胞凋亡模式进行检测分析。结果 3种不同周期特异性细胞凋亡模式在经培养 3、6h的对数放大法分析 ,Sub G1峰均出现在 10 2 荧光道数处 ,且 6h较 3h明显增高。荧光参数对数放大法可避免Sub G1法中Sub G1峰与碎片峰两峰重叠 ,当TdT法清楚地显示凋亡细胞在细胞周期中的时相位置时 ,PC缓冲液的应用则可避免Sub G1峰与G1期峰重叠。结论流式细胞术分析典型细胞凋亡时 ,Sub G1法作定量分析应与对数放大法同步进行 ,才能有效地避免碎片峰与Sub G1峰重叠 ,而PC缓冲液的应用则避免Sub G1峰与G1峰重叠 ,更有效分析细胞凋亡。Sub G1峰应为对称的“高氏分布”图形 ,而不应为“针”形的均一倍性峰。

Reappraisal of the analysis of cell apoptosis with sub G_1 and TdT in flow cytometry

Objective To reappraise the applications of sub G1 and TdT in analyzing apoptosis. Methods Detection of three kinds of different cell cycle specificity apoptosis models were detected by using sub G 1 fluorescence parameter line and logarithms magnifications or TdT. Results Fluorescence parameter logarithms magnifications can prevent the overlay of fractional peak and sub G 1 peak; PC buffer can prevent the overlay of sub G 1 peak and G 1 phase peak during FCM analysis. TdT clearly showed the position of the apoptosis cells in cells cycle. Conclusion When the FCM was used to detect typical apoptosis, quantitative analysis of apoptosis must employ sub G 1 analysis method in combining with DNA gel electrophoresis, however simultaneous quantitative and qualitative analysis must synergically carried out with fluorescence parameter logarithms magnifications. TdT is the best method to detect the position of apoptosis in cell cycle, but quantitative analysis of TdT must be simultaneously conducted with the sub G-1 or other apoptosis qualitative methods. TdT can't be used alone as qualitative means.