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

nal was read on the PHERAstar and the RUBYstar.


Cells were kindly provided by the Institut de Genomique Fonctionnelle (IGF), Montpellier, France and Euroscreen, Gosselie, Belgium. IP-One kit was supplied from Cisbio international (France). The dedicated plate reader RUBYstar and the multimode plate reader PHERAstar were purchased from BMG LABTECH (Germany).

Cell lines (see table 1) expressing the GPCR target of interest were used for measuring IP1 production by stimulation of the ligand. In a 384-well format, the cell suspension was dispensed at 15,000 cells/ 20 L/well. After incubation at 37C, the culture supernatants were completely discarded. Then immediately after, 10 L of stimulation buffer containing various concentrations of ligand were added. After incubation at 37C for 1hr, 5 L IP1-d2 conjugate followed by 5 L of Eu-cryptate labeled anti-IP1 antibody were added. Time-resolved fluorescence at 620 nm and 665 nm were measured with PHERAstar and RUBYstar after incubation at 4C overnight, and the ratios of the signals and Delta F were calculated.

Delta F % = [(Standard or sample Ratio Rationeg ) / Rationeg ] x 100


Results and Discussion
As shown in table 1, IP-One has already been validated on different models and targets - even if the cell lines involved were not specifically optimized for IP-One assay.


The validations also conclusively demonstrate the assays performances in the presence of cell lines using Gα16 or Gqi9 type chimeric constructions. The IP-One assay showed equally good performance on different cellular backgrounds, stable or transient transfected cells, and c
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