Fluorescence lifetime shifts of NAD(P)H during apoptosis measured by time-resolved flow cytometry.
Fluorescence lifetime shifts of NAD(P)H during apoptosis measured by time-resolved flow cytometry.
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DOI：
10.1002/cyto.a.23606
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发表时间：
2019-01
期刊：
影响因子：
--
通讯作者：
Houston JP
中科院分区：
文献类型：
--
作者：
Alturkistany F;Nichani K;Houston KD;Houston JP
来自细胞内代谢物NAD（P）H的自体荧光是一种生物标志物，其被广泛使用并且已知用于可靠地筛选和报告细胞内的代谢活性以及代谢波动。作为一种普遍存在的内源性荧光团，NAD（P）H具有独特的荧光衰减速率，当与辅酶结合时会发生改变。在这项工作中，我们测量了NAD（P）H的荧光衰减或平均荧光寿命（1-3 ns）的变化，并将这种变化与细胞在凋亡过程中经历的代谢变化相关联。我们的测量是用专门设计用于紫外到紫色光谱内的荧光寿命采集的流式细胞仪进行的。我们的方法包括培养、处理和制备细胞用于细胞计数和显微镜测量。我们进行的评价包括观察和定量的内源性发射的变化，由于诱导细胞凋亡，以及通过流式细胞术测量的发射的衰减动力学的变化。早在用细胞凋亡诱导剂处理后15分钟就观察到NAD（P）H荧光寿命的变化。结果还包括相量分析，以评价不同时间点NAD（P）H的游离与结合比。我们将游离与结合比率定义为“短与长”（S/L）荧光寿命的比率，其中发现S/L随着细胞凋亡的增加而一致地降低。通过相量分析等定量框架，NAD（P）H的短寿命和长寿命组分可用于绘制细胞凋亡早期至晚期期间游离和结合NAD（P）H的循环。寿命筛选和相量分析的组合提供了单细胞的高通量代谢谱分析的第一步，并且可以用于筛选和分选，以用于生物医学中的一系列应用。© 2018作者Cytometry Part A由Wiley Periodicals，Inc.出版。代表国际细胞计数促进协会。
Autofluorescence from the intracellular metabolite, NAD(P)H, is a biomarker that is widely used and known to reliably screen and report metabolic activity as well as metabolic fluctuations within cells. As a ubiquitous endogenous fluorophore, NAD(P)H has a unique rate of fluorescence decay that is altered when bound to coenzymes. In this work we measure the shift in the fluorescence decay, or average fluorescence lifetime (1–3 ns), of NAD(P)H and correlate this shift to changes in metabolism that cells undergo during apoptosis. Our measurements are made with a flow cytometer designed specifically for fluorescence lifetime acquisition within the ultraviolet to violet spectrum. Our methods involved culture, treatment, and preparation of cells for cytometry and microscopy measurements. The evaluation we performed included observations and quantification of the changes in endogenous emission owing to the induction of apoptosis as well as changes in the decay kinetics of the emission measured by flow cytometry. Shifts in NAD(P)H fluorescence lifetime were observed as early as 15 min post‐treatment with an apoptosis inducing agent. Results also include a phasor analysis to evaluate free to bound ratios of NAD(P)H at different time points. We defined the free to bound ratios as the ratio of ‘short‐to‐long’ (S/L) fluorescence lifetime, where S/L was found to consistently decrease with an increase in apoptosis. With a quantitative framework such as phasor analysis, the short and long lifetime components of NAD(P)H can be used to map the cycling of free and bound NAD(P)H during the early‐to‐late stages of apoptosis. The combination of lifetime screening and phasor analyses provides the first step in high throughput metabolic profiling of single cells and can be leveraged for screening and sorting for a range of applications in biomedicine. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.
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