CHO-hGPR120 or CHO cells in HBSS (Invitrogen), 20 mM HEPES (Invitrogen), pH 7.4, 0.01% BSA, 1.5 mM 3-isobutyl-1-methylxanthine (Sigma) and d2-labeled cAMP (CisBio) were incubated with GPR120 agonists (1% DMSO) for 45 min at room temperature in white small-volume 384-well plates (Greiner). The reaction was terminated by addition of conjugate and lysis buffer (CisBio) supplemented with anti-cAMP Cryptate (CisBio).
Mouse islets were isolated from pancreas by collagenase digestion, handpicked under microscope following multiple washing steps and dispersed to single cells using TrypLE Express (Gibco). The dispersed islet cells (20 000 cells/well) were incubated with the GPR120 agonists for 30 min in 96-well black plates (Corning) at 37°C in Krebs ringer phosphate HEPES buffer, pH 7.4, with 11 mM Glucose, 0.1% BSA and 0.5 mM 3-isobutyl-1-methylxanthine followed by addition of d2-labeled cAMP and anti-cAMP Cryptate in conjugation and lysis buffer (Cisbio). Produced cAMP was detected with homogenous time resolved fluorescence (HTRF) (λex = 340 nm, λem = 665 and 615 nm) using a Pherastar (BMG Labtech).
Mouse islets were isolated from pancreas by collagenase digestion, handpicked under microscope following multiple washing steps and dispersed to single cells using TrypLE Express (Gibco). The dispersed islet cells (20 000 cells/well) were incubated with the GPR120 agonists for 30 min in 96-well black plates (Corning) at 37°C in Krebs ringer phosphate HEPES buffer, pH 7.4, with 11 mM Glucose, 0.1% BSA and 0.5 mM 3-isobutyl-1-methylxanthine followed by addition of d2-labeled cAMP and anti-cAMP Cryptate in conjugation and lysis buffer (Cisbio). Produced cAMP was detected with homogenous time resolved fluorescence (HTRF) (λex = 340 nm, λem = 665 and 615 nm) using a Pherastar (BMG Labtech).