COS-7 cells were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% dialyzed fetal calf serum (dFCS) and antibiotics. Cells were transiently transfected by electroporation with plasmids encoding HA-tagged 5-HT4R (100 ng/106 cells), then seeded in 96-well plates (16,000 cells/well). Twenty-four hr after transfection, cells were exposed to the indicated concentrations of 5-HT4R ligands in the presence of 0.1 mM of the phosphodiesterase inhibitor RO-20-1724, at 37 °C in 100 µL of HBS (20 mM HEPES; 150 mM NaCl; 4.2 mM KCl; 0.9 mM CaCl2; 0.5 mM MgCl2; 0.1% glucose; 0.1% BSA). After 10 min, cells were then lysed by the addition of the same volume of Triton-X100 (0.1%). Quantification of cAMP production was performed by HTRF® using the cAMP Dynamic kit (Cisbio Bioassays, Codolet, France) according to the manufacturer’s instructions.
Camp dynamic kit
Manufactured by PerkinElmer
Sourced in France
The CAMP Dynamic kit is a laboratory equipment product designed for specific applications. It serves a core function within its intended use. No further details or interpretation can be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
White 384 well plate, by PerkinElmer (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Dulbecco s phosphate buffered saline dpbs, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Forskolin, by Merck Group (1 mentions)
Prism 9, by GraphPad (1 mentions)
Pherastar, by BMG Labtech (1 mentions)
4 protocols using camp dynamic kit
1
5-HT4R Ligand-Induced cAMP Production
2
Measuring cAMP Modulation in Cells
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R3/I5 was obtained from Phoenix Pharmaceuticals (Burlingame, CA, USA). cAMP dynamic kit was bought from Cisbio Bioassays (Codolet, France) and white 384-well plates were from PerkinElmer (Waltham, MA, USA). Forskolin, 3-isobutyl-1-methylxanthine (IBMX), dimethyl sulfoxide (DMSO) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F12), Hank’s balanced salt solution (HBSS), L-glutamine, penicillin-streptomycin, 0.25% trypsin-EDTA, Dulbecco’s phosphate-buffered saline (DPBS) and fetal bovine serum (FBS) were all supplied by Life Technologies (Carlsbad, CA, USA).
3
Chondrocyte cAMP Quantification
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The cAMP concentration of primary chondrocytes was detected by cAMP dynamic kit (Cisbio, France) following the manufacturer’s instructions. cAMP standards were diluted according to the kit instructions. Cells were treated with HL-43 for 2 h and the relative fold change of cAMP was determined by comparing with the cAMP standard curve.
4
cAMP Assay and pH Optimization
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An equal volume (10 μL) of assay buffer at predetermined pH (between pH 3.0 and pH 4.0) was then added to reach desired final pH.
After 30 minutes incubation at room temperature, analytes were detected according to the manufacturer's protocol (cAMP Dynamic kit; CisBio). Fluorescence was read with a PHERAstar plate reader (BMG Labtech, Ortenberg, Germany) using an excitation of 337 nm and emissions of 620 and 665 nm. Raw data were converted to nanomolar cAMP values by interpolation from a cAMP standard curve. The half maximal inhibitory concentration (IC 50 ) and the pH of halfmaximal activation (pH 50 ) determinations were made from an antagonist-or agonist-response curve analysed with a curve fitting program using a four-parameter logistic dose-response equation in GraphPad Prism 9.0 (GraphPad Software, Inc., La Jolla, CA). Data presented are representative of 3 independent experiments performed in quadruplicate for each compound.
Data are represented as averages ± S.D. Data in Table 1 are averages from 3 independent experiments.
After 30 minutes incubation at room temperature, analytes were detected according to the manufacturer's protocol (cAMP Dynamic kit; CisBio). Fluorescence was read with a PHERAstar plate reader (BMG Labtech, Ortenberg, Germany) using an excitation of 337 nm and emissions of 620 and 665 nm. Raw data were converted to nanomolar cAMP values by interpolation from a cAMP standard curve. The half maximal inhibitory concentration (IC 50 ) and the pH of halfmaximal activation (pH 50 ) determinations were made from an antagonist-or agonist-response curve analysed with a curve fitting program using a four-parameter logistic dose-response equation in GraphPad Prism 9.0 (GraphPad Software, Inc., La Jolla, CA). Data presented are representative of 3 independent experiments performed in quadruplicate for each compound.
Data are represented as averages ± S.D. Data in Table 1 are averages from 3 independent experiments.
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