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Label-free monitoring of apoptosis by surface plasmon resonance detection of morphological changes

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Abstract

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.

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Abbreviations

SPR:

Surface plasmon resonance

RVU:

Reflectance variation unit

HBSS:

HEPES buffered salt solution

TRAIL:

TNF (tumor necrosis factor)-related apoptosis-inducing ligand

PE:

Phycoerythrin

7-AAD:

7-aminoactinomycin D

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Acknowledgments

The authors would like to acknowledge Yannick Miron for his technical help with the SPR apparatus and Véronique Blais for her support with flow cytometry analyses. This research was supported by funds from the Canadian Institutes of Health Research (CIHR) and the Natural Sciences and Engineering Research Council of Canada (NSERC) to MG, LG and JBD.

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The authors declare no conflict of interests.

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Correspondence to Michel Grandbois.

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Maltais, JS., Denault, JB., Gendron, L. et al. Label-free monitoring of apoptosis by surface plasmon resonance detection of morphological changes. Apoptosis 17, 916–925 (2012). https://doi.org/10.1007/s10495-012-0737-y

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  • DOI: https://doi.org/10.1007/s10495-012-0737-y

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