Glosensor 22 f vector

Methods for these assays have been previously described^{16 (link)}. In brief, for GTPγs binding, the potency of ligands to stimulate receptor-dependent elevation of the GTPγS-bound Gα_s subunit was determined using membrane preparations from low-receptor-density receptor clonal cell lines. Reactions were incubated for 30 min at room temperature in white, clear-bottom microtiter plates, and per cent of the maximal response was calculated using control wells as a reference. Relative EC₅₀ values were derived by nonlinear regression analysis using the per cent response versus the concentration of ligand and fitted to a four-parameter logistic equation using GraphPad Prism 7 software. For cAMP assays, kinetic cAMP assays were performed in low-density receptor cell clones transfected with the Glosensor 22 F vector (Promega). Cells were equilibrated for 5–20 min and then 20 μl of 10× ligand was added and a luminescence time course was collected. Ligand (10×) was titrated by manual serial dilution in DMSO followed by step-down into assay buffer.

Kinetic cAMP assays were performed in low-density GIPR and GLP-1R cell clones transfected with the Glosensor 22F vector (Promega) (53 (link)). Adherent cells were transiently transfected with Glosensor at 7.5 μg DNA/30 μL Fugene-6 per 150 cm² flask, according to manufacturer’s directions. After 48 hours, cells were removed from flasks using enzyme-free dissociation buffer (13151014, Thermo Fisher Scientific), followed by trituration, brief centrifugation (500g, 5 minutes, room temperature) and filtration (40 μm cell strainer, Thermo Fisher Scientific). Cell viability was quantified with a ViCell (Beckman Coulter) and was typically > 85%. Cells were resuspended at ambient temperature in CO₂-free media (18045088, Thermo Fisher Scientific) containing 0.1 %(v/v) casein (C4765, MilliporeSigma) and 2%(w/v) Glosensor detection reagent (Promega). A total of 18,000 cells per well was plated in 180 μL in solid-bottom, white 96-well plates (3917, Corning). Luminescence was quantified in kinetic mode using a temperature-controlled (22°C) EnVision (PerkinElmer) using standard luminescence settings. Cells were equilibrated for 5–20 minutes, and then 20 μL of 10× ligand was added and a luminescence time course was collected. Ligand (10×) was titrated by manual serial dilution in DMSO followed by step-down into assay buffer.