IP-One assay

We applied the highly sensitive high-throughput homogeneous time-resolved fluorescence (HTRF) technology⁷¹ for detection of IP-One generation using the Cisbio IP-One Tb assay kit, exactly as previously described⁷². In brief, on the day of assay, ligand solutions were prepared in ligand buffer (Hank’s Balanced Salt Solution (HBSS) containing 20 mM HEPES pH 7.4, 1 mM CaCl₂, 1 mM MgCl₂, 40 mM LiCl)) and added to the wells of a 384-well OptiPlate (PerkinElmer, Waltham, USA) in triplicates. When testing for antagonist activity the ligand buffer was supplemented with an EC₈₀ concentration of histamine, and we used a final compound concentration of 20 µM and 2 µM for the first (virtual hits) and second (analogues) screening, respectively. Cell suspensions were added and incubated with ligands for 1 hour, followed by the addition of detection solution (IP-One Tb conjugate & Lysis Buffer +2.5% anti-IP₁ cryptate Tb conjugate +2.5% IP₁-d2 conjugate. 38:1:1) and incubation in the dark for one hour at room temperature. The plate was read on an EnVision 2104 Multilabel Reader (PerkinElmer) by exciting the wells with light of 340 nm and measuring the emission at 615 nm and 665 nm. The fluorescence resonance energy transfer ratios (665 nm/615 nm) were converted to IP₁ concentrations by interpolating values from an IP₁ standard curve generated from an IP₁ calibration stock, provided by the manufacturer (Cisbio).