Label-free monitoring of apoptosis by surface plasmon resonance detection of morphological changes.
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.
Département de Pharmacologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, 3001 12e Avenue Nord, Sherbrooke, QC, J1H 5N4, Canada.
Jean-Sébastien
JS
Maltais
Jean-Bernard
JB
Denault
Louis
L
Gendron
Michel
M
Grandbois
D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D048430:Cell Shape; D002478:Cells, Cultured; D006801:Humans; D020349:Surface Plasmon Resonance; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000494:pharmacology
D017304:Annexin A5; D053221:TNF-Related Apoptosis-Inducing Ligand