Ip one htrf assay kit

Phospholipase C activation was quantified by measuring the inositol monophosphate accumulation in HEK293 cells transiently expressing the mGlu receptors and a chimeric Gqi₉ protein 24 h after transfection with Lipofectamine 2000, as previously described^{35 (link)}. Cells were incubated with the indicated ligands and 10 mM LiCl for 30 min, and the IP1 accumulated was quantified using the IP One HTRF assay kit from Cisbio according to the manufacturer instruction in 384-well plates^{49 (link)}. The amount of cAMP was determined using the Glosensor cAMP assay (Promega Corporation, Madison, USA), as previously described^{26 (link)}. HEK293 cells were co-transfected with the indicated mGluR plasmids, the pGloSensor-22F plasmid and EAAC1 encoding plasmid. The day after, cells were starved for 2 h in serum-free medium and then incubated in Krebs buffer with 450 μg ml⁻¹ luciferin (Sigma-Aldrich) for 30 min, followed by a 30 min incubation with the nanobody at the indicated concentration. The luminescence peak signal was measured on a Mithras microplate reader at 28 °C during 8 min until luminescence signal was stable. Then, forskolin (1 µM) and the indicated concentration of mGlu2 agonist LY379268 were added and luminescence was measured for 30 min.

Cells (2.5 × 10⁴ cells/well) were plated into 96 well trays and cultured overnight. Cells were stimulated with increasing concentration of ligands for 60 min in the presence of LiCl. Samples were lysed and endogenous IP₁ was measured using an IP-One HTRF® assay kit (CisBio) as specified by the manufacturer. Data were normalized to the maximal response elicited by 100 μM of ATP.

cre-luc assays were conducted as described previously (10 (link), 20 (link)). IP₁ accumulation was determined using an IP-One HTRF assay kit (CisBio) and measured using a BMG PHERAstar plate reader with HTRF filters. To monitor Ca²⁺ mobilization, Fluo-4 Direct (Invitrogen) labeling was employed as per the manufacturer's instructions, and time-resolved Ca²⁺ mobilization was measured using confocal microscopy. Briefly, cells were loaded with calcium dye for 30 min at 37 °C followed by incubation at room temperature for a further 30 min. Cells were imaged using a TCS-SP5 confocal microscope (Leica) with a ×20 dry objective. Cells were imaged for ∼1 min before agonist treatment, 10 min after agonist addition, and capturing every 1.2 s. Time-lapse movies were analyzed with the Leica LASAF software.

28–32 h post transfection (XtremeGENE HP) HEK293T cells expressing the indicated sensor were harvested to assess IP₁ levels using the IP-One HTRF assay kit (Cisbio). Cells were gently suspended in their original media, counted using a hemocytometer, and spun down (300 g, 3 min). An appropriate volume of StimB buffer (CisBio: 10 mM Hepes, 1 mM CaCl₂, 0.5 mM MgCl₂, 4.2 mM KCl, 146 mM NaCl, 5.5 mM glucose, 50 mM LiCl, pH 7.4) was added to reach 3 × 10⁶ cells mL⁻¹ density. Cells were incubated at 37 °C for 15 min. The manufacturer’s protocol was modified in order to achieve a high signal to noise ratio. 150 μL of suspension was incubated for one hour with 30 μL of lysis buffer (Cisbio), 54 μL StimB buffer, 6 μL IP₁ conjugated to d2 dye, and 6 μL terbium cryptate-labeled anti-IP₁ monoclonal antibody. IP₁ FRET spectra were collected by exciting samples at 340 nm (bandpass 15 nm). Emission counts were recorded from 600–700 nm (bandpass 10 nm) using a long pass 475 nm filter (FSQ GG475, Newport). Raw IP₁ signal was calculated from the 665 nm to 620 nm ratio. Basal IP₁ signal was corrected by subtracting the untransfected IP₁ ratio from cells expressing transfected sensor. For ligand experiments, data are presented as a change in raw IP₁ ratio following drug treatment. Each experiment had four repeats per condition and was independently repeated at least three times (N > 3).

All peptides were purchased from Mimotopes. Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from Invitrogen. Foetal bovine serum (FBS) was purchased from Thermo Electron Corporation. AlphaScreen reagents, Lance cAMP kit, 384-well Optiplates were purchased from PerkinElmer. SureFire™ ERK1/2 reagents were obtained from TGR Biosciences and PerkinElmer. IP-One HTRF® assay kit was from CisBio. Antibodies were purchased from R&D systems and ThermoFisher. All other reagents were purchased from Sigma-Aldrich or BDH Merck and were of an analytical grade.