Fluorescence lifetime-based discrimination and quantification of cellular DNA and RNA with phase-sensitive flow cytometry
Fluorescence lifetime-based discrimination and quantification of cellular DNA and RNA with phase-sensitive flow cytometry
复制标题
DOI：
10.1002/cyto.a.10022
复制
发表时间：
2003-03-01
期刊：
影响因子：
3.7
通讯作者：
Crissman, HA
中科院分区：
文献类型：
--
作者：
Cui, HH;Valdez, JG;Crissman, HA
背景：同时测量细胞的DNA和RNA含量可为确定细胞的功能状态提供信息，并且在临床上可为各种肿瘤的诊断和分级评估提供信息。目前大多数流式细胞术方法是基于分辨优先结合于任一类型核酸的染料的荧光发射光谱。然而，几种单色核酸结合荧光染料在结合DNA或RNA时在荧光寿命上显示出可分辨的差异。一种荧光探针寿命的差异提供了一种区分一种探针与这些细胞大分子结合并同时测量其细胞含量的替代方法。
方法：选择了三种核酸嵌入染料，碘化丙啶、溴化乙锭和乙锭同二聚体1，以研究它们在结合细胞DNA和RNA时荧光寿命的差异。用特定的核酸酶处理固定的HL - 60细胞，首先确定每种染料在结合细胞DNA、RNA或两者时的寿命值。然后将这些寿命值用作特征来分辨未处理细胞中的细胞DNA和RNA含量。
结果：所有三种染料在结合经核糖核酸酶处理、脱氧核糖核酸酶处理或未处理的细胞时都显示出荧光寿命差异。利用这些寿命值，通过相敏检测从末处理细胞中分辨出DNA、RNA或DNA/RNA的荧光发射。因结合任一类型核酸而产生的寿命差异取决于染料、染色浓度和分析条件。
结论：在不同条件下结合不同大分子时，核酸结合荧光染料的寿命会发生改变。相敏流式细胞术为用一种荧光探针同时区分和定量亚细胞大分子提供了一种独特的方法。数据证明了基于荧光寿命分辨相对细胞DNA和RNA含量的能力。2003年由威利 - 利斯公司出版。
Background: Simultaneous measurement of cellular DNA and RNA content provides information for determination of the functional status of cells and, clinically, for the diagnosis and grading assessment of various tumors. Most current flow cytometric methods are based on resolving the fluorescence emission spectra of dyes that bind preferentially to either type of nucleic acid. However, several monochromatic nucleic acid-binding fluorochromes display resolvable differences in fluorescence lifetime when bound to DNA or RNA. The differences in the lifetime of one fluorescent probe provide an alternate means to distinguish the binding of one probe to these cellular macromolecules and to simultaneously measure their cellular contents.Methods: Three nucleic acid intercalating dyes, propidium iodide, ethidium bromide, and ethidium homodimer 1, were selected to study differences in fluorescence lifetimes when. bound to cellular DNA and RNA. Fixed HL-60 cells were treated with specific nucleases to initially determine the lifetime values of each dye when bound to the cellular DNA, RNA, or both. The lifetime values were then used as the signatures to resolve the cellular DNA and RNA contents in untreated cells.Results: All three dyes showed fluorescence lifetime differences when bound to RNase-treated, DNase-treated, or untreated cells. With these lifetime values, the fluorescence emissions from DNA, RNA, or DNA/RNA were resolved from untreated cells with the use of phase-sensitive detection. The lifetime differences resulting from the binding to either type of nucleic acid depended on the dye, the staining concentration, and the analysis condition.Conclusions: The lifetimes of the nucleic acid-binding fluorochromes were altered when binding to different macromolecules under different conditions. Phase-sensitive flow cytometry provided a unique means for simultaneous discrimination and quantification of subcellular macromolecules with one fluorescent probe. The data demonstrated the capabilities for resolving relative cellular DNA and RNA contents based on fluorescence lifetime. Published 2003 Wiley-liss, Inc.