HEK-293T cells were transfected with cDNAs encoding AT1R, AT2R, or MasR in the presence or the absence of ACE2-HA. Cells were serum-starved in DMEM alone for 2 h before initiating an experiment. Starved cells were detached and suspended in culture medium containing 50 µM of the phosphodiesterase inhibitor, zardaverine (Sigma-Aldrich). Cells were then distributed into 384-well microplates at 2500 cells/well and stimulated for 15 min with increasing concentrations of agonists, including Ang II for AT1R, CGP for AT2R, and Ang 1-7 for MasR. Agonists were added at concentrations 0.1 nM to 3 µM or vehicle alone; this was followed by the addition of 0.5 µM forskolin or vehicle alone for an additional 15 min. Readings were performed after a 1 h incubation at 25 °C. Homogeneous time-resolved fluorescence energy transfer (HTRF) measurements 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).
Pherastar flagship microplate reader
Manufactured by BMG Labtech
Sourced in Germany
The PHERAstar Flagship microplate reader is a high-performance, multi-mode detection platform designed for a wide range of applications in life science research. It offers flexible detection modes, including absorbance, fluorescence intensity, luminescence, and time-resolved fluorescence.
Automatically generated - may contain errors
Lab products found in correlation
Lance ultra camp kit, by PerkinElmer (24 mentions)
Htrf optical module, by BMG Labtech (22 mentions)
Proxiplate 384 well microplates, by PerkinElmer (6 mentions)
Zardaverine, by Bio-Techne (2 mentions)
Camp detection buffer, by PerkinElmer (2 mentions)
Zardaverine, by Merck Group (1 mentions)
Forskolin, by Bio-Techne (1 mentions)
Lance ultra kit, by PerkinElmer (1 mentions)
Eu camp tracer, by PerkinElmer (1 mentions)
Ulight camp monoclonal antibody, by PerkinElmer (1 mentions)
Cgs 21680, by Merck Group (1 mentions)
Forskolin, by Merck Group (1 mentions)
Alphascreen surefire kit, by PerkinElmer (1 mentions)
Enspire multimode plate reader, by PerkinElmer (1 mentions)
P7208 50ug, by Merck Group (1 mentions)
25 protocols using pherastar flagship microplate reader
1
Quantifying GPCR-mediated cAMP Signaling
2
cAMP Quantification via HTRF Assay
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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).
3
Modulating cAMP Responses in Cell Lines
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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 A2AR and MK-801 for NMDAR) or vehicle and stimulated with agonists (CGS-21680 for A2AR 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).
4
Homogeneous FRET Assay for cAMP
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Homogeneous time-resolved fluorescence energy transfer assays were performed using the Lance Ultra cAMP kit (PerkinElmer). HEK-293T cells (1000 per well), growing in medium containing 50 μm zardeverine, were incubated in triplicate for 15 min in white ProxiPlate 384-well microplates (PerkinElmer) at 25°C with vehicle or WIN-55212-2 (100 nm final concentration) before adding vehicle or forskolin (0.5 μm final concentration) and incubating for 15 additional minutes. Fluorescence at 665 nm was analyzed on a PHERAstar Flagship microplate reader equipped with a homogeneous time-resolved fluorescence optical module (BMG Lab Technologies).
5
Quantification of cAMP in HEK-293T Cells
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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).
6
Quantifying cAMP Levels in Cells
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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).
7
Evaluating cAMP Signaling in CB2R and GHSR1a
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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).
8
cAMP Measurement via HTRF Assay
9
Measuring cAMP in HEK-293T cells
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Signaling experiments have been performed as previously described (Navarro et al., 2010 (link), 2016 (link), 2018b (link); Hinz et al., 2018 (link)). Two hours before initiating the experiment, HEK-293T cell-culture medium was replaced by serum-starved DMEM medium. Then, cells were detached, resuspended in growing medium containing 50 μM zardaverine and placed in 384-well microplates (2,500 cells/well). Cells were pretreated (15 min) with CBG -or vehicle- and stimulated with agonists (15 min) before adding 0.5 μM forskolin or vehicle. Readings were performed after 15 min incubation at 25°C. HTRF energy transfer measures were performed using the Lance Ultra cAMP kit (PerkinElmer, Waltham, MA, United States). Fluorescence at 665 nm was analyzed in a PHERAstar Flagship microplate reader equipped with an HTRF optical module (BMG Lab Technologies, Offenburg, Germany).
10
cAMP Signaling Assay in Cells
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Two hours before initiating the experiment, neuron culture medium was replaced by serum-starved DMEM medium. Then, cells were detached and resuspended in serum-starved DMEM medium containing 50 μM zardaverine, 0.1% BSA and 5mM HEPES. Cells were plated in 384-well microplates (2,000 cells/well), pretreated (15 min) with the corresponding antagonists -or vehicle- and stimulated with agonists (15 min) before adding 0.5 μM forskolin (15 min). Readings were performed after 1 h incubation at 25°C. Homogeneous time-resolved fluorescence energy transfer (HTRF) measures were performed using the Lance Ultra cAMP kit (PerkinElmer, Waltham, MA, USA). According to the manufacturer and to our previous experience phenol red in DMEM does not affect cAMP concentration determinations. Fluorescence at 665 nm was analyzed on a PHERAstar Flagship microplate reader equipped with an HTRF optical module (BMG Lab technologies, Offenburg, Germany).
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