The cAMP levels in cultured C688 cells were determined using a cAMP-Glo Max Assay (Promega, USA), according to the manufacturer’s protocol. Cells were grown in tissue culture treated 96-well, white plates with clear, flat bottoms (Brand, Germany). To each well, 103 C688 cells were added and cultured overnight under standard conditions. The next day, the medium was discarded and a mixture of 2.5 mg/mL plant extract or gallic acid and 25 nM CTX (2.125 μg/mL) in DMEM supplemented with 30 mM MgCl2 was added to each well. The plates were incubated for 2 h at 37 °C in 5% CO2. Next, the proper amount of detection solution and kinase glo reagent were added. The luminescence (RLU) was measured using a SpectraMax M5e plate reader. As controls, cells were incubated with only: plant extracts, CTX, or DMEM. The cAMP level was calculated as a change in RLU (ΔRLU) for the sample incubated with only plant extract/gallic acid/DMEM and the sample incubated with a mixture of plant extract/gallic acid/DMEM and CTX. Each sample was tested in three repeats.
Camp glo max assay
Manufactured by Promega
Sourced in United States
The CAMP-Glo Max Assay is a luminescent-based assay that detects and quantifies cyclic adenosine monophosphate (cAMP) levels in cells. The assay utilizes a thermostable luciferase enzyme to produce a luminescent signal proportional to the cAMP concentration in the sample.
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Lab products found in correlation
3 isobutyl 1 methyl xanthine (ibmx), by Merck Group (3 mentions)
Fluostar optima, by BMG Labtech (2 mentions)
Ro 20 1724, by Merck Group (2 mentions)
Biotek synergy h1 microplate reader, by Agilent Technologies (1 mentions)
Synergy h1 plate reader, by Agilent Technologies (1 mentions)
Cl316 243, by Bio-Techne (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Estradiol, by Merck Group (1 mentions)
Indomethacin, by Merck Group (1 mentions)
Forskolin, by Merck Group (1 mentions)
Sr59230a, by Merck Group (1 mentions)
Fulvestrant, by Merck Group (1 mentions)
Amphotericin b, by Merck Group (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Propranolol, by Cayman Chemical (1 mentions)
Cl316, by Merck Group (1 mentions)
Synergy h1, by Agilent Technologies (1 mentions)
10 protocols using camp glo max assay
1
cAMP Measurement in C688 Cells
2
Intracellular cAMP Assay in GLUTag Cells
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The intracellular cAMP levels were measured using a cAMP-Glo™ Max Assay (Promega, Madison, WI, USA) kit according to the manufacturer’s protocol. GLUTag cells were seeded in a white 96-well plate at an initial density of 10,000 cells per well. The following day, the media were removed and replaced with the complete induction buffer (low-glucose DMEM containing 100 μM Ro 20–1724 and 500 μM IBMX) and vehicle control (0.05% DMSO (v/v)) or 2.5 μM 18:1-LPA with or without 10 μM Ki16425 for 2 h at 37 °C, 5% CO2. Cells were then lysed with the cAMP Detection Solution for 20 min at room temperature. Finally, 50 μL Kinase-Glo® Reagent was added to each well and incubated for 10 min at room temperature before measuring luminescence using a BioTek Synergy™ H1 microplate reader (Agilent Technologies). The cAMP level was normalized to vehicle-treated control cells.
3
Measuring Intracellular cAMP in C2C12 Cells
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Transduced mock (IRES-GFP) and Calcr (CalcR-C1a-IRES-GFP) C2C12 cells were isolated by FACS based on GFP expression and seeded on 0.1% gelatin-coated, white culture 96-well plates (Falcon, 353296) at 3x103 cells/well. After overnight culture, the cells were incubated with the complete induction medium containing DMEM/1%PS/500μM IBMX (isobutyl-1-methylxanthine; Sigma, 17018)/100µM Ro 20-1724 ([4-(3-butoxy-4-methoxy-benzyl) imidazolidone]); Sigma, B8279)/MgCl2 40mM, collagen, solvant HOAc or Elcatonin (0.1U/ml) for 3h. The amount of intracellular cAMP was measured using cAMP-Glo Max Assay (Promega, V1681) following the manufacturer’s protocol. Luminescence was quantified with FLUOstar OPTIMA (BMG Labtech). EC50 value was determined with GraphPad Prism software using a sigmoid dose-response curve (variable slope).
4
cAMP Quantification in IL-1β-Treated Cells
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The cAMP-Glo Max Assay (Promega, cat. #V1681) was followed according to the manufacturer’s instructions. Briefly, H322 cells were seeded at a density of 10,000 cells/well in 96-well clear bottom assay plate (Corning, cat. #3610). The next day, cells were stimulated with 10 ng/mL IL-1B (n = 7) or vehicle control (n = 8) for 8 h in serum-free media according to the manufacturer’s instructions. An 8 h time point was chosen based upon data in human myometrial cells showing that IL-1B increases cAMP levels through a prostaglandin E2 dependent pathway after 5 h of IL-1B treatment that peaks at 12 h (Oger et al., 2002 (link)). A standard curve was constructed according to the manufacturer’s instructions. The plate was read on an Agilent Bio Tek Syngery LX multi-mode reader using endpoint luminescence protocol. The cAMP concentrations for vehicle and IL-1B-treated H322 cells were calculated using the values from the standard curve and a sigmoidal 4PL curve (GraphPad Prism 9).
5
Measuring Intracellular cAMP in C2C12 Cells
6
cAMP Assay in N/TERT-1 Keratinocytes
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N/TERT-1 keratinocytes were plated in to 96 well plates at 8000 cells/well and grown to 80% confluence. On the day of the cAMP assay the adherent cells were treated with PBS-IBMX buffer (100 μM IBMX + 0.4 mM CaCl2) for 30 min to inactivate phosphodiesterase. The induction buffer (PBS + 20 mM MgCl2) was used to dilute test compounds at different concentrations (agonist, Forskolin and TAMRA control). Cells were treated in 40 μl of induction buffer with relevant test compounds for 30 min at 37°C. 10 μl cAMP detection solution (buffer with enzyme PKA) was added to cells and incubated for 20 min. Cell lysates (50 μl) were transferred into a white-bottom 96-well plate (Greiner Bio-One GMBH, Frickenhausen, Germany). After addition of 50 μl Kinase-Glo reagent reaction was performed for 10 min before measuring luminescence using BioTek Synergy™ H1 plate reader (BioTek; Winooski, VT, U.S.A.). All the procedures were followed according to Promega cAMP-Glo™ Max Assay (Madison, WI, U.S.A.).
7
Isolation and Differentiation of Adipose Stromal Cells
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Stromal vascular (SV) fraction was obtained from subcutaneous adipose depots of two-month old mice as described.8 (link),20 (link),21 (link) Isolated SV cells were cultured in DMEM supplemented with 10% FBS, 100 units/mL penicillin, 100 mg/mL streptomycin and 25 ng/mL Amphotericin B (Sigma). White adipogenesis was induced as described.8 (link),21 (link) Beige adipogenesis was induced similarly, except for an addition of 5 nM indomethacin (Sigma) and 2 nM T3 (Sigma).20 (link) Fulvestrant (1 µM, Sigma), Estradiol (1 µM, Sigma), CL-316,243 (Tocris Bioscience), SR59230A (1 µM, Sigma), Propranolol (1 µM, Cayman Chemical, Ann Arbor, MI) or vehicles (ethanol, H2O or DMSO in equivalent dilutions) were added to confluent cells with each media change. To induce thermogenic genes, cells were treated with 10 µM Forskolin (Sigma), 1 µM Norepinephrine (dissolved in H2O with 0.125 mM ascorbic acid, Sigma) or 1 µM CL-316,243–8 hr for RNA isolation or 24 hr for immunostaining. “Sub-optimal beige media” - IBMX and Dexamethasone (Sigma) were removed. Oil Red O staining, Nile Red staining and immunostaining were done as described.8 (link),21 (link) Relative changes in cAMP levels were measured using cAMP-Glo™ Max Assay (Promega, Madison, WI) following induction in PBS and IBMX (500 µM, Sigma) with or without 10 µM Forskolin for 15 min.
8
Measuring cAMP Levels in Keratinocytes
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Keratinocytes were cultured to confluence in 96-well culture plates and then treated with 3PPP (1 mM) or ethanol (control) with or without addition of SQ-22536 (100 μM) or L-cis-diltiazem (100 μM) dissolved in medium for 15 min at 37°C. cAMP level in the cells was analyzed by using cAMP-Glo™ Max Assay (Promega, Wisconsin, United States of America) according to the manufacturer’s instructions. The luminescence was detected with a microplate reader (SYNERGY H1, BioTek, Vermont, United States of America).
9
Pharmacological Modulation of cAMP in HEK-293T Cells
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Untransfected HEK-293T cells or those transiently expressing either pCMV6 empty vector, pCMV6-DRD1-WT or pCMV6-DRD1-T37K were cultured on white, clear-bottom 96-well plates for 24 h. Cells were briefly washed with PBS to remove traces of serum and then incubated with concentrations of Chloro APB, pramipexole, apomorphine, rotigotine or bromocriptine in PBS that contained 100 µM IBMX and 100 µM Ro20-1724 for 30 min at 37 °C. cAMP levels were then recorded using the cAMP Glo Max assay (Promega) according to the manufacturer’s instructions. Briefly, cells were lysed with the cAMP detection solution plus protein kinase A for 20 min at room temperature. An equal volume of Kinase-Glo was added and incubated for 10 min at room temperature, and plates were read after 10 min using a HidEx Sense luminometer. For analysis, values were inverted for clear graphical representation of cAMP production. Comparisons were conducted using a two-way ANOVA with multiple comparisons. Statistical significance was considered where p < 0.05.
10
cAMP Quantification in IL-13 Stimulated Cells
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The cAMP-Glo Max Assay (Promega, cat. #V1681) was followed according to the manufacturer’s instructions and as previously described by our lab (Sponchiado et al., 2023 (link)). Briefly, NCI-H322 cells were seeded at a density of 10,000 cells/well in 96-well clear bottom assay plate per the manufacturer’s protocol (Corning, cat. #3610). The next day, cells were stimulated with IL-13 or vehicle control (as detailed above). A standard curve was constructed according to the manufacturer’s instructions. The plate was read on an Agilent Bio Tek Syngery LX multi-mode reader using endpoint luminescence protocol. The cAMP concentrations for vehicle and IL-13-treated NCI-H322 cells were calculated using the values from the standard curve and plotted in a sigmoidal 4PL curve (GraphPad Prism 9) as we previously described (Sponchiado et al., 2023 (link)).
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