Htrf optical module

HEK-293T cell growth medium was replaced by serum-starved DMEM medium 2 h prior to the experiment. Next, cells were detached, suspended in growing medium containing 50 µM zardaverine and plated in 384-well microplates (2,500 cells/well), pretreated (15 min) with vehicle and stimulated with the receptor agonist (CGS21680, 15 min) before adding 0.5 µM forskolin or vehicle. Readings were performed after 1h incubation at 25 °C. Homogeneous time-resolved fluorescence energy transfer (HTRF) measures were performed 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).

Homogeneous time-resolved fluorescence energy transfer (HTRF) assays were performed using the Lance Ultra cAMP kit (PerkinElmer), based on competitive displacement of a europium chelate-labeled cAMP tracer bound to a specific antibody conjugated to acceptor beads. We first established the optimal cell density for an appropriate fluorescent signal. This was done by measuring the TR-FRET signal as a function of forskolin concentration using different cell densities. The forskolin dose-response curves were related to the cAMP standard curve, to establish which cell density provides a response that covers most of the dynamic range of the cAMP standard curve. Cells (1000–2000 HEK-293T or 4000 to 5000 primary cultures per well) growing in medium containing 50 μM zardeverine were pre-treated with toxins or the corresponding vehicle in white ProxiPlate 384-well microplates (PerkinElmer) at 25 °C for the indicated time and stimulated with agonists for 15 min before adding 0.5 μM forskolin or vehicle and incubating for an additional 15 min period. Fluorescence at 665 nm was analyzed on a PHERAstar Flagship microplate reader equipped with an HTRF optical module (BMGLab technologies, Offenburg, Germany).

Two hours before initiating the experiment, culture medium for HEK-293T-transfected or primary neuronal or glial cells was exchanged by serum-starved DMEM medium. Then, the cells were detached, resuspended in a growing medium containing 50 µM zardaverine (Tocris Bioscience, Bristol, UK) and plated in 384-well microplates (2500 cells/well), pretreated (15 min) with the corresponding antagonists (SCH-58261 for A_2AR and MK-801 for NMDAR) or vehicle and stimulated with agonists (CGS-21680 for A_2AR and NMDA for NMDAR) (15 min) before adding 0.5 μM forskolin or vehicle (15 min). The readings were performed after a 1 h incubation (room temperature). Homogeneous time-resolved fluorescence energy transfer (HTRF) measures were performed 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).

cAMP was determined using the Lance Ultra cAMP kit (PerkinElmer), which is based on homogeneous time-resolved fluorescence energy transfer technology. Briefly, HEK-293T cells (1000 per well), growing in medium containing 50 µM zardaverine (Tocris #1046) as phosphodiesterase inhibitor, were incubated for 15 min in white ProxiPlate 384-well microplates (PerkinElmer) at 25 °C with vehicle (DMSO, 0.1% v/v final concentration) WIN-55,212-2 or CP55,940 (doses ranging from 0.0025 to 1 µM final concentration) before adding vehicle (DMSO, 0.1% v/v final concentration) or forskolin (Tocris, Bristol, UK, #1099, 0.5 μM final concentration) and incubating for 15 additional min. Every condition was assayed in triplicate within each individual experiment. Fluorescence at 665 nm was analysed on a PHERAstar Flagship microplate reader equipped with an HTRF optical module (BMG Lab Technologies, Offenburg, Germany).

The ad hoc LanceUltra kit (PerkinElmer, Waltham, MA, USA) was used for cAMP determination using homogenous assays. Transfected HEK-293T cells or primary neurons were seeded in 6-well plates. Two hours before initiating the experiment, culture medium was substituted by non-supplemented DMEM medium. After detachment, cells were re-suspended in non-supplemented medium containing 50 μM zardaverine. Cells were pretreated (30 min) with 200 nM CBD, 200 nM CBG, or vehicle and, 5 min later, stimulated with selective agonists. Forskolin (0.5 μM) or vehicle were then added for a period of 15 min. Finally, the reaction was stopped by the addition of the Eu-cAMP tracer and the ULight-cAMP monoclonal antibody prepared in the “cAMP detection buffer” of the LanceUltra kit. All steps were performed in 384-well microplates at 25 °C. Then, 60 min later, homogeneous time-resolved fluorescence energy transfer (HTRF) measures were obtained in a PHERAstar Flagship microplate reader equipped with an HTRF optical module (BMGLab 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).

Two hours before initiating the experiment, HEK-293T or neuronal cell-culture medium was exchanged to serum-starved DMEM or neurobasal medium, as corresponds. Then, cells were detached, resuspended in the serum-starved medium containing 50 µM zardaverine, plated in 384-well microplates (2500 cells/well), pre-treated (15 min) with the corresponding antagonists or the vehicle, and then stimulated with agonists (15 min) before adding 0.5 μM forskolin or vehicle. Readings were performed after 1 h incubation at 25 °C. Homogeneous time-resolved fluorescence energy transfer (HTRF) measures were obtained using the Lance Ultra cAMP kit (PerkinElmer, Waltham, MA, USA) [62 (link)]. Fluorescence at 665 nm was analyzed on a PHERAstar Flagship microplate reader equipped with an HTRF optical module (BMG Lab technologies, Offenburg, Germany).