Ip one tb htrf kit

Inositol phosphate assay was performed using the IP-One-Tb HTRF kit (Cisbio Bioassays, France) following the manufacturer’s instructions. CHO-M1 cells were transfected with PKR1 or PKR2 plus either empty vector or MRAP2 at a ratio of 1:10 (receptor to MRAP2). The day before the experiment, the transfected cells were plated in a white 384-well plate at a density of 20,000 cells/well. The next day the cells were treated with different concentrations of PK1, PK2 or 1 μM carbachol in stimulation buffer containing lithium for 1 hr at 37°C, and then incubated with IP1-d2 conjugate and Anti-IP1 cryptate Tb in lysis buffer for 1 hr at room temperature. HTRF signal was read at 615 nm and 665 nm using a Spectramax I3 plate reader equipped with the HTRF Cisbio cassette. For each condition, signal obtained with 1 μM carbachol, an agonist of the M1 muscarinic receptor, was used for normalization. Results were then normalized to the highest signal from the control (receptor + empty vector).

Inositol phosphate (IP) accumulation was measured using a homogenous time-resolved FRET (HTRF) assay (HTRF IP-One Tb kit, Cisbio Bioassays, Codolet, France). HEK293T cells were transiently cotransfected with FLAG-hGPR35-eYFP and the G-protein chimaera Gα_q/135 (a form of Gα_q in which the C-terminal 5 amino acids were replaced with the corresponding pentapeptide from Gα₁₃) using PEI. After 16-24 h of incubation at 37°C in a 5% CO₂ humidified atmosphere, the cells were resuspended in IP-One stimulation buffer (10 mM HEPES, 1 mM CaCl₂, 0.5 mM MgCl₂, 4.2 mM KCl, 146 mM NaCl, 5.5 mM glucose and 50 mM LiCl, pH7.4) and seeded at 10,000 cells/well in white, solid-bottom, 384-well plates. Ligands were diluted in IP-One stimulation buffer according to the manufacturer's instructions. Antagonist compounds were preincubated with cells for 15 min at 37°C prior to the addition of the agonist. Cells were incubated with ligand(s) for 2 h at 37°C, before the addition of d₂-conjugated IP monophosphate (IP₁; 3 μl/well) and anti-IP₁ Lumi4™-Tb cryptate (3 μl/well) diluted in lysis buffer. After incubation at room temperature for 1 h, HTRF was measured using a PHERAstar FS plate reader (BMG-Labtech). IP₁ accumulation was measured by the fluorescence ratio of 665 nm/620 nm.

HEK 293 T cells were seeded into T75 flasks at 90,000 cells/cm² and incubated at 37 °C and 5% CO₂ overnight. The following day cells were transiently transfected for 24 hours. IP₁ accumulation was measured using Homogeneous Time-Resolved Fluorescence (HTRF) IP-ONE Tb Kit (Cisbio, Codolet, France) as per manufacturer’s instruction. The assay was perfomed using 10,000 cells/well into white, low volume 384-well plates (Greiner Bio-One) The HTRF signal was read on an Envision imager (Perkin-Elmer) after a 1 hour room temperature incubation.