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Introduction
Time-resolved fluorescence resonance energy transfer(TR-FRET) has become a popular technique in the field ofhigh-throughput screening. Its popularity is mainly due to thehigh sensitivity¹, the lack of any radioactive reagents and thehealth and safety issues these cause. TR-FRET is based onthe transfer of photons between a lanthanide complex, thedonor to a suitable acceptor, when they are in close proximity.The lanthanide donor complex exhibits a long fluorescencelifetime with a shallow signal decay curve. When this donorcomplex is excited by a pulsed light source, e.g. flash lamp orfluorometer laser, its extremely long lifetime allows the separationof this signal from the light emitted by other fluorophores witha normal, shorter lifetime (Fig. 1). Together with the largeStoke shift of the lanthanides fluorescence and the ratiometricnature of the readout, interference from false-positive arisingfrom autofluorescent compounds in the screening collection isdrastically reduced².

Amongst the different commercial TR-FRET kits availableCisbio technology is one of the most popular³. The HTRF(homogeneous time-resolved fluorescence) technology isdedicated to high-throughput drug screening and leadgeneration and has been extensively used as a tool forscreening a number of tyrosine kinases alongside measuringother molecular complexes.

The latest development from Cisbio is the introduction of theIP-One HTRF assay, a cell-based functional assay for themonitoring of phospholipase C coupled (PLC) receptors andthe validation of the function of various G-protein coupledreceptors (GPCRs)⁴. G-protein coupled receptors (GPCRs) area large protein family of transmembrane receptors that reactwith specific extracellular mole cules, activate inside signaltransduction pathways and, ultimately, cellular responses.

G-protein coupled receptors are involved in many diseases andare the target of about 25 % of the world top selling drugs.They also represent approximately one third of the number oftargets currently being investigated by the pharmaceuticalindustry⁵.

GPCRs can be classified into 3 subclasses based on thesecondary messenger they affect. Gαs or Gαi coupled GPCRsregulate cAMP levels, while Gq coupled GPCRs activatephospholipase C (PLC) and trigger the inositol phosphate (IP)cascade (Fig. 2).

Several metabolites in this pathway, including IP3, haveextremely short half lives, making them difficult to accuratelyquantify. IP-One, a downstream metabolite of IP3,accumulates in cells following Gq receptor activation and isstable in the presence of LiCl inhibiting the conversion of IP1into myoinositol by inositol monophosphatase⁴.

Material and Methods
Assay technology
G-protein coupled receptor screening was established basedon the IP-One HTRF technology (Cisbio, France). The IP-OneHTRF assay is based on a monoclonal antibody, specific toIP1, labelled with europium cryptate which competes withboth native IP1 produced by cells and IP1 coupled with theHTRF acceptor (d2). The specific signal (energy transfer) isinversely proportional to the concentration of IP1 in thecalibrator or cell lysate (Fig. 2).

The experiment was conducted following the recommendedassay protocol (Fig. 3).

Tissue culture
Cells were cultured according to standard procedures in175 cm² tissue culture flasks (Cat.-No. 660 175, Greiner Bio-One GmbH, Frickenhausen, Germany) in Earle MEM medium(Biochrom AG, Berlin, Germany) containing 10 % fetal horseserum, 2 % glutamine, 10 ml non essential amino acids (50 x,Biochrom AG, Berlin, Germany), pyruvate (Biochrom AG, Berlin,Germany) in a humidified atmosphere at 5 % CO₂ and 37.

Cultures were split (1:10) every 4 days using standard trypsinationprocedures (0.05 % trypsin containing 0.02 % EDTAsolutions from Biochrom AG, Berlin, Germany). Medium waschanged after 2 days.

For the preparation of the assay plates cells were grown to80 % confluence and concentrated in cell culture media in a50 ml polypropylene tube (Cat.-No. 227 261, Greiner Bio-OneGmbH, Frickenhausen, Germany) to a final concentration of1,000 cells / .

Preparation of assay plates
The validation of the IP-One cell based assay was carried outin white cell culture treated microplates from four differentmanufacturers. Before using these microplates backgroundwas determined under standard HTRF conditions. The excitationwavelength used was 337 nm with emission wavelengthsread at 665 and 620 nm in a BMG time-resolved fluorescencereader (Pherastar, BMG LabTech, Offenburg, Germany). Themeasurement at 620 nm represents the background of theassay whereas the results at 665 nm represent the timeresolvedfluorescence of the acceptor.

Each well that was used in the assay was seeded at 40,000cells / well (assay volume of 40 ) and the plates were incubatedovernight at 37 allowing the cells to attach to the cellculture treated surfaces.

The following steps, including all controls, were conductedaccording to the protocol supplied by Cisbio (Fig. 3).

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