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These systems incorporate an optical biosensor into a micro-titre plate to measure the cellular response to a stimulus. Specifically, the systems are able to detect changes in mass at the sensor surface resultant from alterations in cell shape and/or protein recruitment or migration to/from the membrane.
The technology is generic being independent of signaling pathway, it is non-invasive (no molecular biology required) and generates a kinetic response over seconds, minutes or hours resulting in an information rich readout.
Label-free techniques may also be of benefit where there is no simple/standard biological readout e.g. for de-orphanisation of receptors and identification of pharmacological “tool” compounds.
The Aurelia Bioscience team have developed assays for a number of 7 Transmembrane Receptors (7TMR) (or GPCR) targets, conducting high throughput screens and mechanistic studies. Specifically…….
Screening: Utilised the Corning Epic system to screen a number of 7-TMR targets including agonist screens for two dual coupled receptors (Gi/Gq and a Gs/Gi couples)
Published proof of concept: Comparing the output from label-free and calcium flux readouts for a muscarininc receptor antagonist screen in a peer reviewed journal. (Ref: Journal of Receptors and Signal Transduction, 2009; 29(3–4): 163–172)
100,000 compounds were screened in antagonist mode using a CHO cells expressing a muscarinic receptor.
Inhibition data generated from the label-free assay was compared to data for the same compounds tested in a calcium flux screen using FLIPR.
The correlation of the two sets of data highlighted a group of “hit” compounds that were unique to the label-free assay (Set C)
A recombinant cell line was challenged with compound and the label-free response measured over a period of 15 minutes to determine agonism.
The cells were then stimulated with a standard agonist at an EC80 concentration and any antagonist effect measured over the next 45 minutes.
Circles highlight data points used for XC50 calculation.
Neutrophils were isolated from human donor whole blood using density gradient centrifugation and plated into 384 well biosensor plate.
IL-8 was added to wells at different doses (shown) and responses measured over a 25 minute period.
The instrument is able to measure label-free responses in addition to fluorescent and luminescent responses sequentially from the same wells of a plate.
It can therefore be used to multiplex complex cell-based assays measuring kinetic responses over seconds, minutes or hours.
- Receptor tyrosine kinases e.g. EGFR
- Ion channels e.g. Potasssium channels
- Viral proliferation in cells – cell death
- Cell adhesion – or prevention of
- Cell proliferation – apoptosis
If you want to understand more about your cellular target by applying a label-free approach then...