Label-free monitoring of apoptosis by surface plasmon resonance detection of morphological changes
Label-free monitoring of apoptosis by surface plasmon resonance detection of morphological changes
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DOI：
10.1007/s10495-012-0737-y
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发表时间：
2012-08-01
期刊：
影响因子：
7.2
通讯作者：
Grandbois, Michel
中科院分区：
文献类型：
--
作者：
Maltais, Jean-Sebastien;Denault, Jean-Bernard;Grandbois, Michel
凋亡可以通过生物分子标记物（如TUNEL和膜联蛋白V）或荧光半胱天冬酶底物进行常规表征。细胞凋亡也可以用显微镜半定量表征，其目标是形态学特征，如细胞变圆、核凝聚和破碎以及细胞膜起泡。这种无标签的方法为这些事件的演变提供了有限的分辨率，并且严重依赖于形态学特征的主观识别。在这里，我们提出了一种基于表面等离子体共振（SPR）检测内皮细胞模型中由于细胞凋亡诱导而发生的微小形态学变化的无标记分析（EA.hy926）。首先，膜联蛋白V检测证实我们的细胞模型在12小时内对TRAIL有反应。然后，我们表明SPR可以通过测量(1)凋亡信号被启动者半胱天冬酶整合并传递给执行者半胱天冬酶的潜伏期的持续时间，(2)死亡底物被切割和形态学变化发生的执行阶段的速率，以及(3)凋亡的总程度来精确监测细胞凋亡。利用这些参数，我们表征了TRAIL （EA.hy926, HeLa, AD-293）和抗fas抗体（HeLa）对外源性途径的反应，以及紫外线暴露（HeLa）对内在途径的反应。通过比较细胞凋亡的SPR时间过程和相对比显微照片，我们发现细胞凋亡的形态学特征是SPR信号的主要贡献者。总之，我们的研究结果验证了SPR作为一种准确的无标记检测方法用于实时监测凋亡引发的细胞形态变化。
Apoptosis can be routinely characterized using biomolecular markers such as in the TUNEL and the annexin V assays or by using fluorescent caspase substrates. Apoptosis can also be semi-quantitatively characterized using microscopy, which targets morphological features such as cell rounding, nuclear condensation and fragmentation as well as cell membrane blebbing. This label-free approach provides a limited resolution for the evolution of these events in time and relies heavily on subjective identification of the morphological features. Here we propose a label-free assay based on surface plasmon resonance (SPR) detection of minute morphology changes occurring as a result of apoptosis induction in an endothelial cell model (EA.hy926). At first, annexin V assays confirmed that our cellular model was responsive to TRAIL over a 12-hour period. Then, we show that SPR allows accurate monitoring of apoptosis by measuring (1) the duration of the latency period during which the apoptotic signal is integrated by the initiator caspases and transmitted to the executioner caspases, (2) the rate of the execution phase in which death substrates are cleaved and morphological changes occur, and (3) the total extent of apoptosis. Using these parameters, we characterized the responses obtained with TRAIL (EA.hy926, HeLa, AD-293) and the anti-Fas antibody (HeLa) for the extrinsic pathways and UV exposure (HeLa) for the intrinsic pathways. By comparing the SPR time-course of apoptosis with phase contrast micrographs, we demonstrate that the cell morphological hallmarks of apoptosis are the major contributors to the SPR signal. Altogether, our results validate the use of SPR as an accurate label-free assay for the real-time monitoring of apoptosis-triggered cell morphological changes.