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

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.

iRAGE as a novel carboxymethylated peptide that prevents advanced glycation end product-induced apoptosis and endoplasmic reticulum stress in vascular smooth muscle cells

“…The custom-built SPR apparatus has been described in details in previous publications 44,45 and was applied by our group for the monitoring of apoptosis in a realtime manner 46 . SPR measurements were done by measuring the reflectance at a fixed angle in the quasi-linear section of the angular scan (i.e.…”

“…First, surface plasmon resonance (SPR) was used as a label-free assay to follow apoptosis in real-time 46 . Figure 2 shows that A7r5 cells display a robust reflectance decline starting 4 hours after stimulation with CML-HSA, reaching 75.6 ± 2.8% of the maximum possible decrease after 20 hours.…”

Label-Free Monitoring ofμ-Opioid Receptor–Mediated Signaling

“…Gold substrates were prepared and surface plasmon resonance (SPR) experiments were conducted as previously described (Cuerrier et al, 2008;Maltais et al, 2012). The HEK FlagMOP cells were grown to confluence on a gold substrate surface precoated with a solution of 5 mg/ml fibronectin (Sigma-Aldrich) and 200 mg/ml gelatin (BD, Franklin Lakes, NJ).…”

“…For example, Stallaert et al (2012) used an impedance-based system to assess functional selectivity at the b 2 adrenergic receptor, whereas Morse et al (2011Morse et al ( , 2013 used an optical-based system to assess opioid receptor signaling. We previously showed that SPR represents a valuable label-free technique to monitor GPCR signaling and apoptosis (Cuerrier et al, 2008;Chabot et al, 2009;Maltais et al, 2012).…”

A two-compartment microfluidic device for long-term live cell detection based on surface plasmon resonance

“…Because of the advantage of sensitive, real-time, and label-free, SPR cell detection has drawn much attention. [20][21][22] It has been proved that cellular morphologic change, 23,24 cell-substrate interactions, 25,26 and even the ligand-induced intracellular signaling events 27,28 can be monitored by SPR in real time. Recently, SPR has been combined with other techniques, such as electrochemical measurement, nanoparticles, and microscope imaging, for multi-parametric cell profiling.…”