Glosensor camp assay

Manufactured by Promega

Sourced in United States

The GloSensor cAMP assay is a bioluminescent-based kit designed to detect and quantify levels of cyclic AMP (cAMP) in live cells. It utilizes a genetically-encoded biosensor that produces a luminescent signal in the presence of cAMP, allowing real-time monitoring of cAMP changes in a cellular environment.

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Lab products found in correlation

Glosensor camp reagent, by Promega (4 mentions) Pglosensor 22f camp plasmid, by Promega (3 mentions) Glomax 20 20n luminometer, by Promega (3 mentions) White 96 well plate, by Corning (3 mentions) Co2 independent media, by Thermo Fisher Scientific (2 mentions) Glosensor camp reagent, by Thermo Fisher Scientific (2 mentions) Sucrose, by Merck Group (2 mentions) Co2 independent medium, by Thermo Fisher Scientific (2 mentions) Pglosensor22f camp, by Promega (2 mentions) Forskolin, by Merck Group (2 mentions) Luciferin, by Merck Group (2 mentions) Prism 7, by GraphPad (2 mentions) Flexstation 3, by Molecular Devices (1 mentions) Softmax pro software, by Molecular Devices (1 mentions) Prism 10, by GraphPad (1 mentions) Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions) Synergy h1 microplate reader, by Agilent Technologies (1 mentions) Tryple express enzyme 1x, by Thermo Fisher Scientific (1 mentions) Hek293 cell, by American Type Culture Collection (1 mentions) Ip one htrf assay kit, by PerkinElmer (1 mentions)

35 protocols using glosensor camp assay

GloSensor cAMP Assay for Cell-Based Analysis

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Example 10

GloSensor cAMP assay

The GloSensor cAMP assay was conducted per manufacturer's protocol (Promega). In brief, ChoK1 human GLP-1R/GIPR-expressing cells were seeded at 15,000 cells per well in a 96 well plate and incubated for 16 hours in a humidified 37° C., 5% CO₂incubator. Cells were transiently transfected with 100 ng pGloSensor −22F cAMP plasmid using FuGENE HD for 24 hours. Cells were then equilibrated with 0.1% BSA, 2% GloSensor cAMP reagent in CO₂independent media (Invitrogen) for 2 hours at room temperature. Cells were preincubated for 15 min with or without 0.43 M sucrose (Sigma). Basal luminescence measurements were taken for 10 minutes prior to addition of 1248 in conditions with or without sucrose. Immediately following addition of 1248, kinetic luminescence was measured using an integration time of 0.1-1 second every 60 seconds. Data were analyzed using GraphPad Prism software and presented in FIG. 26.

US10905772B2. Method of treating or ameliorating metabolic disorders using GLP-1 receptor agonists conjugated to antagonists for gastric inhibitory peptide receptor (GIPR) (2021-02-02). AMGEN INC. [US]. Inventors: Yuan Cheng [US], Chawita Netirojjanakul [US], Jerry Ryan Holder [US], Bin Wu [US], James R. Falsey [US], Bradley J. Herberich [US], Kelvin Sham [US], Leslie P. Miranda [US], Shu-Chen Lu [US], Murielle M. Veniant-Ellison [US], Shanaka Stanislaus [US], Junming Yie [US], Jing Xu [US].

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HTRF-based Intracellular cAMP Assay

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MEG-01 or other indicated cell types, seeded in white walled, clear bottom 96-well plate were stimulated with test substances in Hanks’ balanced salt solution (HBSS) including 1.8 mM CaCl₂ and 10 mM glucose for 15 min at 37°C. Intracellular cAMP concentrations were determined by using an homogenous time-resolved fluorescence (HTRF)-based cAMP assay (Cisbio) following manufacturer’s instructions.
In case of heterologously expressed receptors, intracellular cAMP levels were measured by coexpression of a plasmid encoding a cytosolic cAMP-sensitive bioluminescent probe (GloSensor^™ cAMP Assay, Promega). Forty-eight hours after transfection of receptors and cAMP probe, cells were removed from the incubator and medium was changed to HBSS containing 1.8 mM CaCl₂, 10 mM glucose and 2% (v/v) of GloSensor^™ cAMP reagent. Two hours after of incubation in dark at room temperature, intracellular cAMP was estimated in the “kinetic” mode by continuously recording the light produced over indicated time following ligand stimulation. For “end-point” determination, cells were stimulated with indicated ligands, and light generated was measured 15 minutes later with an integration time of 1250 ms. In both cases, light was recorded by a 96-well plate reader (Flexstation 3, Molecular Devices) and results were evaluated by SoftMaxPro software (Molecular Devices).

Tunaru S., Chennupati R., Nüsing R.M, & Offermanns S. (2016). Arachidonic Acid Metabolite 19(S)-HETE Induces Vasorelaxation and Platelet Inhibition by Activating Prostacyclin (IP) Receptor. PLoS ONE, 11(9), e0163633.

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Measuring HCA2/3 Signaling Inhibition

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The inhibitory effects of different HCA2 and HCA3 constructs or mutants on forskolin-induced cAMP accumulation were measured using the GloSensor cAMP assay (Promega). Expi293 cells were transiently co-transfected with the GloSensor and various mutants of HCA2 plasmids using PEI in 6-well plates. After incubation at 37 °C for 24 h, the transfected cells were harvested and washed with Dulbecco’s phosphate-buffered saline (PBS), centrifuged at 190×g for 5 min, and suspended in HBSS (Thermo Fisher Scientific) containing 0.01% BSA and 5 mM HEPES (pH 7.4). The cells were then resuspended in 2% GloSensor cAMP reagent (Promega) at room temperature for 2 h. The cell suspension was seeded into a 96-well white plate (Corning) at a volume of 80 μL per well. A 10-μL volume of 10 x drug buffer diluted in HBSS containing HEPES and 0.01% BSA was added to each well; the plates were incubated for 10 min. Then, a 10-μL volume of 10-uM forskolin (Sigma-Aldrich) (final 1 μM) was added and the plates were incubated for 20 min at room temperature. The luminescence of the cells was measured using a SpectraMax i3x multi-plate reader. Data were analyzed using “Sigmodal, 4PL, X is concentration” in GraphPad Prism 10.0.

Suzuki S., Tanaka K., Nishikawa K., Suzuki H., Oshima A, & Fujiyoshi Y. (2023). Structural basis of hydroxycarboxylic acid receptor signaling mechanisms through ligand binding. Nature Communications, 14, 5899.

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Measuring Intracellular cAMP Levels

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The levels of the intracellular 3′, 5′-cyclic AMP (cAMP) were measured by the GloSensor cAMP assay (Promega) as described in the previous studies^{28 (link),41 (link)} which has been widely used. The human full-length SSTRs and mutants were cloned into the pcDNA3.1+ vector with a HA signal sequence followed by a Flag tag at the N-terminus. HEK293 cells (ATCC, CRL-1573) were seeded into 6-well microplates at a density of 1.0 × 10⁶ cells/well and cultured for 24 h at 37 °C with 5% CO₂. Cells were then co-transfected with SSTR wild-type or mutants and GloSensor plasmids. After 24 h of transfection, the cells were dissociated with TrypLE™ Express Enzyme (1X) (Thermo Fisher Scientific) and collected by centrifugation at 500 × g for 5 min. Then the collected cells were suspended in Hank’s balanced salt solution [HBSS (Thermo Fisher Scientific)] supplemented with D-fluorescein potassium salt solution (YEASEN), and then the suspension cells were seeded into 96-well plates with 90 μl/well. After incubating at room temperature for 40 min, 5 μM forskolin and ligands diluted in HBSS were added to each well and the plates were incubated at room temperature for 15 min. Fluorescence signals were measured using the Synergy H1 microplate reader (BioTek) and the data presented are means of at least three independent experiments.

Zhao J., Fu H., Yu J., Hong W., Tian X., Qi J., Sun S., Zhao C., Wu C., Xu Z., Cheng L., Chai R., Yan W., Wei X, & Shao Z. (2023). Prospect of acromegaly therapy: molecular mechanism of clinical drugs octreotide and paltusotine. Nature Communications, 14, 962.

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Quantification of mGluR-Mediated Signaling

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Phospholipase C activation was quantified by measuring the inositol monophosphate accumulation in HEK293 cells transiently expressing the mGlu receptors and a chimeric Gqi₉ protein 24 h after transfection with Lipofectamine 2000, as previously described^{35 (link)}. Cells were incubated with the indicated ligands and 10 mM LiCl for 30 min, and the IP1 accumulated was quantified using the IP One HTRF assay kit from Cisbio according to the manufacturer instruction in 384-well plates^{49 (link)}. The amount of cAMP was determined using the Glosensor cAMP assay (Promega Corporation, Madison, USA), as previously described^{26 (link)}. HEK293 cells were co-transfected with the indicated mGluR plasmids, the pGloSensor-22F plasmid and EAAC1 encoding plasmid. The day after, cells were starved for 2 h in serum-free medium and then incubated in Krebs buffer with 450 μg ml⁻¹ luciferin (Sigma-Aldrich) for 30 min, followed by a 30 min incubation with the nanobody at the indicated concentration. The luminescence peak signal was measured on a Mithras microplate reader at 28 °C during 8 min until luminescence signal was stable. Then, forskolin (1 µM) and the indicated concentration of mGlu2 agonist LY379268 were added and luminescence was measured for 30 min.

Scholler P., Nevoltris D., de Bundel D., Bossi S., Moreno-Delgado D., Rovira X., Møller T.C., El Moustaine D., Mathieu M., Blanc E., McLean H., Dupuis E., Mathis G., Trinquet E., Daniel H., Valjent E., Baty D., Chames P., Rondard P, & Pin J.P. (2017). Allosteric nanobodies uncover a role of hippocampal mGlu2 receptor homodimers in contextual fear consolidation. Nature Communications, 8, 1967.

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Measuring cAMP Response of Lamprey bPPL

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The changes in the intracellular cAMP concentration of pigment-expressing HEK293S cells were measured using the GloSensor cAMP assay (Promega), as previously reported^{13 (link),29 (link)}. Briefly, cDNAs of lamprey bPPL were tagged with the epitope sequence for the monoclonal Rho1D4 antibody (ETSQVAPA). Tagged cDNA was inserted into the plasmid vector, pcDNA3.1 (Invitrogen). The expression constructs for bPPL were co-transfected with the pGloSensor-22F cAMP plasmid (Promega), and the transfected cells were incubated overnight in culture medium containing 10% fetal bovine serum (FBS) with 11-cis-retinal. Before the measurement, culture medium was replaced with a CO₂-independent medium containing 10% FBS and 2% GloSensor cAMP Reagent (Promega). After equilibration with the medium and obtention of a steady basal signal, the cells were treated with 3.5 μM forskolin, a direct activator of adenylyl cyclase, to increase the intracellular cAMP levels. Luminescence, representing the amount of cAMP, was measured at 25 °C using a GloMax 20/20n Luminometer (Promega). To measure the light-induced change in cAMP levels, the transfected cells were irradiated with blue LED light for 5 sec. The relative response curve of lamprey bPPL was analysed using the GloSensor cAMP assay without forskolin following the previously described method^{29 (link)}.

Kawano-Yamashita E., Koyanagi M., Wada S., Saito T., Sugihara T., Tamotsu S, & Terakita A. (2020). The non-visual opsins expressed in deep brain neurons projecting to the retina in lampreys. Scientific Reports, 10, 9669.

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Quantifying cAMP Signaling in Cells

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cAMP production was determined using the Promega GloSensor cAMP assay (Promega) (Binkowski et al. 2011 (link)). Briefly, cells were seeded (20,000 cells/well) in white 96-well clear-bottomed microplates. The next day, the cells were transfected with pGloSensorTM-22F cAMP plasmid (150 ng) encoding an engineered cAMP-sensitive luciferase, using Lipofectamine LTX (Invitrogen, Cergy-Pontoise, France) according to the manufacturer’s instructions. Twenty-four hours after transfection, the medium was removed, and the cells were incubated for 2 hr at 20°C in 90 μL of the equilibration medium, a substrate-containing medium (GloSensor^TM cAMP reagent) diluted to 6% in DMEM containing 10% FCS. The cells were incubated with p,p′-DDT, p,p′-DDE, or hormones for 30 min, and end-point luminescence was recorded on a Synergy^TM 2 microplate luminometer (Biotek). Graphs were fitted to the data using GraphPad Prism 6 (GraphPad Software, Inc.), and the results are expressed as the mean ± SEM from at least three independent experiments performed in triplicate. Concentration–response data were fitted using a four-parameter equation.

Munier M., Grouleff J., Gourdin L., Fauchard M., Chantreau V., Henrion D., Coutant R., Schiøtt B., Chabbert M, & Rodien P. (2016). In Vitro Effects of the Endocrine Disruptor p,p’-DDT on Human Follitropin Receptor. Environmental Health Perspectives, 124(7), 991-999.

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Transient Transfection of TAAR1 Variants

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HEK293 cells were maintained in Minimum Essential Media (MEM, Biochrom GmbH, Berlin, Germany) supplemented with 5% fetal calf serum (FCS) and non-essential amino acids (Biochrom AG, Berlin, Germany), in humidified air at 37°C and 5% CO2. Cells were seeded in poly-L-lysine coated (Biochrom GmbH, Berlin, Germany) 96-well assay plates, at a density of 1.5 × 10⁵ cells/ml. After 24 h, cells were transiently transfected using METAFECTENE (0.45 μl/well), in supplement-free Advanced MEM (Life Technologies, Carlsbad, CA, USA). For the HiBiT assay, cells were transfected with the pcDNA3 empty vector (mock), TAAR1-WT, or co-transfected with TAAR1-WT and variants to resemble the heterozygous state, as well as GLPR as a positive control. For the GloSensor^™ cAMP assay, cells were co-transfected with TAAR1-WT and variants and the GloSensor plasmid F22 (Promega, Mannheim, Germany). As a negative control, TAAR1 was exchanged with empty vector (mock). In order to mimic the heterozygous state of the variants, TAAR1-WT and mutants were transfected in equimolar plasmid amounts.

Rutigliano G., Bräunig J., Del Grande C., Carnicelli V., Masci I., Merlino S., Kleinau G., Tessieri L., Pardossi S., Paisdzior S., Dell’Osso L., Biebermann H, & Zucchi R. (2019). Non-Functional Trace Amine-Associated Receptor 1 Variants in Patients With Mental Disorders. Frontiers in Pharmacology, 10, 1027.

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GloSensor cAMP Assay for GPR3 Activation

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The GloSensor cAMP assay was performed according to the instructions of Promega. AD293 cells were split into 6-well plate (300,000 cells/mL) and then transfected with the pGloSensor™-22F cAMP plasmid and pcDNA3-GPR3 plasmid by Lipofectamine 2000, at a ratio of 2:1 (μL:μg). 24 h after transfection, cells were split into 96-well plate for another 24 h incubation. After removal of cell medium, 90 μL of 3% v/v (the GloSensor cAMP reagent vs serum-free medium) substrate was added into each well. After 2 h incubation at RT, background measurements were taken, and then 10 μL 10× stock compounds were added for the second measurements of ligand activation via EnVision 2105 (PerkinElmer). Relative cAMP level was calculated by the cAMP level after addition of ligand divided by the cAMP level before addition of ligand (basal level). Stock FAs (i.e., 10–50 mM) were solubilized in DMSO, then diluted in cell culture buffer to designated concentrations and sonicated to ensure that they were evenly distributed in the buffer before being added to cells.

Xiong Y., Xu Z., Li X., Wang Y., Zhao J., Wang N., Duan Y., Xia R., Han Z., Qian Y., Liang J., Zhang A., Guo C., Inoue A., Xia Y., Chen Z, & He Y. (2024). Identification of oleic acid as an endogenous ligand of GPR3. Cell Research, 34(3), 232-244.

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Quantifying Intracellular cAMP Levels

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The GloSensor™ cAMP Assay (Promega) was used for detecting changes in the intracellular levels of cAMP. Initially, a Greiner Bio-One 96 Well Plate (white, TC treated) was seeded at 8000 cells per 100 μl well, as described above. After 24 h, the cells were transfected with the pGloSensor™ cAMP Plasmid using the Lipofectamine3000 transfection reagent as above. Between 24 and 48 h later, allowing for the accumulation of the biosensor, the medium in the 96-well plate was changed to CO₂-independent medium supplemented with GlutaMAX™ (Life Technologies), 10% foetal bovine serum and 2% GloSensor™ cAMP Reagent. The plate was then stored for 2 h at room temperature to equilibrate. Following the incubation period, the cells were treated with relevant drugs and their luminescence values were measured after 20 min in the BMG FLUOstar OPTIMA Microplate Reader, with forskolin as a positive control, and DMSO as a negative control.

Jagannath A., Varga N., Dallmann R., Rando G., Gosselin P., Ebrahimjee F., Taylor L., Mosneagu D., Stefaniak J., Walsh S., Palumaa T., Di Pretoro S., Sanghani H., Wakaf Z., Churchill G.C., Galione A., Peirson S.N., Boison D., Brown S.A., Foster R.G, & Vasudevan S.R. (2021). Adenosine integrates light and sleep signalling for the regulation of circadian timing in mice. Nature Communications, 12, 2113.

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