Camp detection buffer

HEK-293T cells transfected with the cDNAs for CB₁R (1 μg) and/or GHS-R1a (1.5 μg) and neuronal primary cultures were plated in 6-well plates. Two hours before initiating the experiment, the cell-culture medium was replaced by the non-supplemented DMEM medium. Then, cells were detached, resuspended in the non-supplemented DMEM medium containing 50 μM zardaverine, and plated in 384-well microplates (2,500 cells/well). Cells were pretreated (15 min) with the corresponding antagonists (1 μM rimonabant for CB₁R and 1 μM YIL 781 for GHS-R1a) or vehicle and stimulated with agonists (1 nM, 10 nM, 100 nM, and 1 μM ACEA for CB₁R or 200 nM ghrelin for GHS-R1a; 15 min) before the addition of 0.5 μM FK or vehicle. Finally, the reaction was stopped by the addition of the Eu-cAMP tracer and the ULight-conjugated anti-cAMP monoclonal antibody prepared in the “cAMP detection buffer” (PerkinElmer). All steps were performed at 25°. Homogeneous time-resolved fluorescence energy transfer (HTRF) measures were performed after 60 min incubation at RT using the Lance Ultra cAMP kit (PerkinElmer, Waltham, MA, USA). Fluorescence at 665 nm was analyzed on a PHERAstar Flagship microplate reader equipped with an HTRF optical module (BMG Lab Technologies, Offenburg, Germany).

Microglial and neuronal primary cultures were plated in 6-well plates. Two hours before initiating the experiment, cell-culture medium was replaced by non-supplemented DMEM medium. Then, cells were detached, resuspended in non-supplemented DMEM medium containing 50 μM zardaverine, and plated in 384-well microplates (2500 cells/well). Cells were pretreated (15 min) with the corresponding antagonists (1 µM SCH 58261 for A_2AR and 1 µM PSB 10 for A₃R) or vehicle and stimulated with agonists (100 nM PSB 777 for A_2AR and 100 nM 2-Cl-IB-MECA for A₃R) (15 min) before the addition of 0.5 μM FK or vehicle. Finally, reaction was stopped by addition of the Eu-cAMP tracer and the ULight-cAMP monoclonal antibody prepared in the “cAMP detection buffer” (PerkinElmer). All steps were performed at 25°. Homogeneous time-resolved fluorescence energy transfer (HTRF) measures were performed after 60 min of incubation at RT using the Lance Ultra cAMP kit (PerkinElmer, Waltham, MA, USA). Fluorescence at 665 nm was analyzed on a PHERAstar Flagship microplate reader equipped with an HTRF optical module (BMG Lab Technologies, Offenburg, Germany).

HEK-293T cells transfected with the cDNAs for CB₂R (0.5 µg) and/or GHSR1a (1 µg) and neuronal primary cultures were plated in 6 well plates. Two hours before initiating the experiment, neuronal culture or HEK-293T cell-culture media were exchanged to non-supplemented DMEM medium. Then, cells were detached, re-suspended in non-supplemented medium containing 50 µM zardaverine, and plated in 384-well microplates (2500 cells/well). Cells were pretreated (15 min) with the corresponding antagonists (1 µM AM 630 for CB₂R or 1 µM YIL 781 for GHSR1a) or vehicle and stimulated with agonists (200 nM JWH-133 for CB₂R or 200 nM ghrelin for GHSR1a) (15 min) before the addition of 0.5 μM forskolin or vehicle. Finally, reaction was stopped by addition of the Eu-cAMP tracer and the ULight-cAMP monoclonal antibody prepared in the “cAMP detection buffer” (PerkinElmer). All steps were performed at 25º. Homogeneous time-resolved fluorescence energy transfer (HTRF) measures were performed after 60 min incubation using the Lance Ultra cAMP kit (PerkinElmer, Waltham, MA, USA). Fluorescence at 665 nm was analyzed on a PHERAstar Flagship microplate reader equipped with an HTRF optical module (BMG Lab technologies, Offenburg, Germany).