Alphascreen surefire kit

To determine ERK1/2 phosphorylation, 40,000 HEK-293T cells/well or 50,000 neurons/well were plated in transparent Deltalab 96-well plates and kept in the incubator for 48 h. The medium was substituted by serum-free DMEM medium for 2–4 h before initiating the experiment. Then, HEK-293T cells and striatal neurons were pretreated for 10 min at 25°C with vehicle, PB-28 (300 nM) or cocaine (30 μM) followed by the addition of 200 nM SKF-81297, the D₁R specific agonist. 10 min after activation, cells/neurons were placed on ice and washed twice with cold PBS before the addition of 30 μl of lysis buffer for 15 min. Supernatants (10 μl) were placed in white ProxiPlate 384-well microplates, and ERK1/2 phosphorylation was determined using the AlphaScreen^®SureFire^® kit (Perkin Elmer) and the EnSpire^® Multimode Plate Reader (PerkinElmer, Waltham, MA, United States).

To determine extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation, 40,000 HEK-293T cells transfected with cDNA for AT₁R (1 μg) and/or AT₂R (1 μg) or 50,000 striatal neurons or glial cells primary cultures were plated in each well of transparent Deltalab 96-well microplates. Two hours before the experiment, the medium was substituted by serum-starved DMEM medium. Then, cells were treated or not for 15 min with the selective antagonists Candesartan or PD123319 in serum starved DMEM medium followed by 7-min treatment with the selective agonists Ang II and/or CGP-42112A. Cells were then washed twice with cold PBS before the addition of lysis buffer (15-min treatment). Ten microliters of each supernatant were placed in white ProxiPlate 384-well microplates and ERK1/2 phosphorylation was determined using AlphaScreen®SureFire® kit (Perkin Elmer) following the instructions of the supplier and using an EnSpire® Multimode Plate Reader (PerkinElmer, Waltham, MA, USA).

Hippocampal neurons, microglial cells, or HEK-293T cells cotransfected with the cDNA for the protomers of the NMDA receptor, GluN1 (1 μg) and GluN2B (0.75 μg), and/or with the cDNA for CB₂R (1 μg) were plated in transparent Deltalab 96-well microplates. Primary microglial cells were activated by incubating cells with 1 μM LPS and 200 U/mL IFN-γ during 48 h. Two hours before the experiment, the medium was substituted by serum-starved DMEM medium. Cells were treated or not for 10 min with the selective antagonists (MK-801 (1 μM) or SR-144528 (1 μM)) followed by 7 min treatment with the selective agonists (NMDA (15 μM) and/or JWH-133 (100 nM)). Cells were then washed twice with cold PBS before the addition of lysis buffer (15 min treatment). Ten microliters of each supernatant was placed in white ProxiPlate 384-well microplates and ERK1/2 phosphorylation was determined using an AlphaScreen®SureFire® kit (Perkin Elmer) following the instructions of the supplier and using an EnSpire® Multimode Plate Reader (PerkinElmer).

To determine ERK1/2 phosphorylation, 40,000 transfected HEK-293T cells/well, 50,000 neurons/well or 40,000 astroglia cells/well were plated in transparent Deltalab 96-well microplates and kept in the incubator for 48 h (transfected HEK-293T cells) or 10 days (astroglial and neuronal primary cultures). Then, primary cultures were transfected with siRNA for NR2A, siRNA for NR2B or vehicle. Then, neurons were treated with Aβ1-42 (1 µM) or vehicle for 48 h while astroglia was treated with 1 μM LPS plus 200 U/mL IFN-γ for 48 h. Two to four hours before initiating the experiment, the medium was substituted by a serum-starved DMEM medium. Then, cells were pre-treated at 25 °C for 10 min with the specific antagonist MK-801 or vehicle in serum-starved DMEM medium and stimulated for an additional 7 min with increasing concentrations of NMDA or vehicle. Cells were then washed twice with cold PBS before the addition of lysis buffer (20 min treatment in constant agitation). An amount of 10 μL of each supernatant was placed in white ProxiPlate 384-well microplates and ERK 1/2 phosphorylation was determined using AlphaScreen^®SureFire^® kit (Perkin Elmer) following the instructions of the supplier and using an EnSpire^® Multimode Plate Reader (PerkinElmer, Waltham, MA, USA).

To determine the ERK1/2 phosphorylation, 40,000 HEK-293T cells/well, 50,000 microglia cells/well or 50,000 neurons/well were plated in transparent 96-well microplates and kept in the incubator for 48 h (HEK-293T cells) or 12 days (microglia and neuronal culture cells). Two to four h before initiating the experiment, the medium was substituted for a serum-starved DMEM medium. Then, the cells were pre-treated at room temperature for 10 min with the specific antagonists (SCH-58261 for A_2AR and MK-801 for NMDAR) or vehicle in a serum-starved DMEM medium and stimulated for an additional 10 min with the specific agonists (CGS-21680 for A_2AR and N-methyl d-aspartate (NMDA) for NMDAR) or vehicle. The cells were then washed twice with cold PBS before the addition of a lysis buffer (20 min treatment in constant agitation). Subsequently, 10 μL of each supernatant was placed in white ProxiPlate 384-well microplates and the ERK 1/2 phosphorylation was determined using the AlphaScreen^®SureFire^® kit (Perkin Elmer, Waltham, MA, USA) following the instructions of the supplier and using an EnSpire^® Multimode Plate Reader (PerkinElmer, Waltham, MA, USA).

To determine ERK1/2 phosphorylation, 40,000 HEK-293T-CB₂R cells/well were plated in transparent Deltalab 96-well microplates and kept at the incubator for 24 h. Two to four hours before the experiment, the medium was replaced by serum-free DMEM. Then, cells were treated with 100 nM JWH133 and increasing concentrations of CBD in serum-free medium at 25°C for 7 min. Cells were then washed twice with cold PBS before addition of lysis buffer (20 min treatment). Ten microliters of each supernatant were placed in white ProxiPlate 384-well microplates and ERK1/2 phosphorylation was determined using AlphaScreen^®SureFire^® kit (PerkinElmer) following the instructions of the supplier and using an EnSpire^® Multimode Plate Reader (PerkinElmer, Waltham, MA, United States).