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HTRF IP-One Assay Performed on the PHERAstar and RUBYstar Plate Readers

vertheless does not possess the necessary profile for an HTS tool because of its overlybrief physiological existence. Moreover, there is no inhibitor available for the phosphatases which convert IP3 into IP2 and then into IP1. On the other hand, the final stage of the cascade when IP1 is transformed into myo-inositol can be blocked with the use of LiCl, which enables one of the Gq pathways essential sub-products to be stabilized (figure 1), just as IBMX action prevents cAMP degradation. In its final configuration, it brings into play a highly specific MAb coupled to europium cryptate and the IP1 conjugated to the new HTRF acceptor, d2. This tiny proprietary molecule, with photophysical characteristics similar to those of XL665 (the reference HTRF acceptor), improves the technologys performances markedly, particularly in terms of IC50 stability and of measurement dynamics.


Materials and Methods
Cisbios homogeneous IP-One assay can be carried out in a single microplate, into which the cells have been dispensed the day before the actual test is run (figure 2). The cell stimulation conditions meet the particular characteristics of the cell line used - generally 30 minutes at 37C. Quantification of the accumulated IP1 is obtained after dispensing the two diluted conjugates into the lysis solution. Measurements can then start to be taken after just one hour of incubation, and be repeated as many times as necessary without impacting final data (e.g. IC50). The standard curve for the kit was run according to the package insert protocol in white 384-well plates (COSTAR cat# 3711384) with 20 L total assay volume. The HTRF sig
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