Sailer B L, Nastasi A J, Valdez J G, Steinkamp J A, Crissman H A
Life Sciences Division, Los Alamos National Laboratory, NM 87545, USA.
Cytometry. 1996 Oct 1;25(2):164-72. doi: 10.1002/(SICI)1097-0320(19961001)25:2<164::AID-CYTO5>3.0.CO;2-H.
Deuterium oxide (D2O) has been shown in previous studies to increase both the fluorescence lifetime and fluorescence intensity of propidium iodide (PI) and ethidium bromide (EB) when bound to nucleic acid structures. We have used spectroscopic analysis and conventional and phase-sensitive flow cytometry to compare changes in PI and EB fluorescence intensity and lifetime bound to DNA and fixed Chinese hamster ovary (CHO) cells in the presence of D2O vs. phosphate-buffered saline (PBS). Spectroscopic and flow cytometric studies showed a twofold enhancement of fluorescence intensity of PI and EB bound to fixed CHO cells in D2O and a 5 ns increase in PI and EB fluorescence lifetimes in D2O. The fluorescence lifetime of HL-60 cells stained with PI or EB was found to be 1-2 ns different from that of CHO cells, indicating that the lifetime of these fluorochromes is sensitive to chromatin configuration in different cells types. Apoptotic subpopulations of HL-60 cells had a significantly reduced fluorescence lifetime compared to nonapoptotic subpopulations. Results indicate that different chromatin states, or differences in the structures of PI and EB, lead to alterations in the fluorescence intensity and fluorescence lifetime of these intercalating probes.
在先前的研究中已表明，重水（D2O）与核酸结构结合时，会增加碘化丙啶（PI）和溴化乙锭（EB）的荧光寿命及荧光强度。我们运用光谱分析以及传统和相敏流式细胞术，比较了在重水与磷酸缓冲盐水（PBS）存在的情况下，PI和EB与DNA及固定的中国仓鼠卵巢（CHO）细胞结合时荧光强度和寿命的变化。光谱和流式细胞术研究表明，在重水中与固定的CHO细胞结合的PI和EB的荧光强度增强了两倍，且在重水中PI和EB的荧光寿命增加了5纳秒。发现用PI或EB染色的HL-60细胞的荧光寿命与CHO细胞的荧光寿命相差1 - 2纳秒，这表明这些荧光染料的寿命对不同细胞类型中的染色质构型敏感。与非凋亡亚群相比，HL-60细胞的凋亡亚群的荧光寿命显著降低。结果表明，不同的染色质状态或PI和EB结构的差异，会导致这些嵌入探针的荧光强度和荧光寿命发生改变。
