Evaluation of Caspase-3 Activity During Apoptosis with Fluorescence Lifetime-Based Cytometry Measurements and Phasor Analyses.
Evaluation of Caspase-3 Activity During Apoptosis with Fluorescence Lifetime-Based Cytometry Measurements and Phasor Analyses.
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DOI：
10.1002/cyto.a.24207
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发表时间：
2020-12
期刊：
影响因子：
--
通讯作者：
Houston JP
中科院分区：
文献类型：
--
作者：
Nichani K;Li J;Suzuki M;Houston JP
Caspase‐3是一种被很好地描述的蛋白酶，具有许多影响细胞命运的作用。在细胞凋亡过程中，caspase‐3作为一个刽子手caspase，具有重要的蛋白水解功能，导致程序性细胞死亡的最后阶段。由于这一关键作用，caspase‐3被利用作为细胞内控制细胞凋亡的靶点来治疗结果。然而，凋亡过程中caspase‐3的激活受到其他作用和功能（如旁分泌信号）的挑战。这篇简短的报告介绍了一种使用流式细胞仪测量激发态荧光寿命和信号处理方法来跟踪caspase‐3水平的方法，该方法可导致基于图形相量的解释。本试验采用已建立的Förster共振能量转移（FRET）生物探针；在细胞凋亡诱导过程中，连接的供体和受体荧光团可被caspase‐3切割。通过细胞间的衰变动力学数据和相量分析，我们生成了caspase激活轨迹，用于解释凋亡过程中的激活。当基于寿命的细胞术与FRET生物探针和相量分析相结合时，酶的激活可以通过相位和调制数据来简化和量化。我们设想将这种方法外推到高含量筛选中，并通过细胞分析数据加强相量方法的力量。当细胞内荧光探针用作酶活性传感器时，这些分析可用于通过细胞的相位和调制“寿命指纹”来聚集细胞。©2020作者。《细胞术》A部分由Wiley期刊有限责任公司代表国际细胞术进步协会出版。
Caspase‐3 is a well‐described protease with many roles that impact the fate of a cell. During apoptosis, caspase‐3 acts as an executioner caspase with important proteolytic functions that lead to the final stages of programmed cell death. Owing to this key role, caspase‐3 is exploited intracellularly as a target of control of apoptosis for therapeutic outcomes. Yet the activation of caspase‐3 during apoptosis is challenged by other roles and functions (e.g., paracrine signaling). This brief report presents a way to track caspase‐3 levels using a flow cytometer that measures excited state fluorescence lifetimes and a signal processing approach that leads to a graphical phasor‐based interpretation. An established Förster resonance energy transfer (FRET) bioprobe was used for this test; the connected donor and acceptor fluorophore is cleavable by caspase‐3 during apoptosis induction. With the cell‐by‐cell decay kinetic data and phasor analyses we generate a caspase activation trajectory, which is used to interpret activation throughout apoptosis. When lifetime‐based cytometry is combined with a FRET bioprobe and phasor analyses, enzyme activation can be simplified and quantified with phase and modulation data. We envision extrapolating this approach to high content screening, and reinforce the power of phasor approaches with cytometric data. Analyses such as these can be used to cluster cells by their phase and modulation “lifetime fingerprint” when the intracellular fluorescent probe is utilized as a sensor of enzyme activity. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.
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DOI：
10.18632/aging.100459
发表时间：
2012-05
期刊：
Aging
影响因子：
--
作者：
Favaloro B;Allocati N;Graziano V;Di Ilio C;De Laurenzi V
通讯作者：
De Laurenzi V
影响因子：
3.7
作者：
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通讯作者：
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影响因子：
2.5
作者：
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通讯作者：
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影响因子：
82.9
作者：
通讯作者：
--
影响因子：
2.1
作者：
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