Measurements of cAMP production were performed using homogeneous time resolved fluorescence (HTRF) cAMP dynamic kits (CisBio, Codolet, France). All buffers and reagents were according to manufacturer’s protocol. In short, 0.1 μl of compounds in DMSO were added to small-volume 384-well plates (Greiner, Frickenhausen, Germany). 12,000 (5 μl) cells in Hank’s balanced salt solution (HBSS) (Life Technologies) and 20 mM HEPES (pH7.4; Life Technologies) were added per well. The production of cAMP was stimulated for 75 min at 37°C by addition of 5 μl 3-isobutyl-1-methylxanthine (0.375 mM final concentration; Sigma-Aldrich, St Louis, MO) and ZnCl2 (0 to 30 μM; Sigma). The reaction was stopped by addition of 5 μl cAMP-d2 and 5 μl anti-cAMP cryptate. The cAMP production was detected by HTRF (λex = 340 nm, λem = 665 and 615 nm) using a Pherastar (BMG Labtech, Ortenberg, Germany).
Htrf camp dynamic kit
Manufactured by PerkinElmer
Sourced in France
The HTRF-cAMP dynamic kit is a tool used for the detection and measurement of cyclic adenosine monophosphate (cAMP) levels in various biological samples. It employs an homogeneous time-resolved fluorescence (HTRF) technology to provide quantitative results.
Automatically generated - may contain errors
Lab products found in correlation
384 well plate, by Greiner (1 mentions)
Hepes ph 7, by Thermo Fisher Scientific (1 mentions)
Zncl2, by Merck Group (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
3 isobutyl 1 methylxanthine, by Merck Group (1 mentions)
Pherastar, by BMG Labtech (1 mentions)
Cb17 scid mice, by Charles River Laboratories (1 mentions)
3 isobutyl 1 methylxanthine ibmx, by Cayman Chemical (1 mentions)
Adrenocorticotropic hormone acth, by Merck Group (1 mentions)
Mithras lb940 reader, by Berthold Technologies (1 mentions)
Htrf ip1 kit, by PerkinElmer (1 mentions)
Proxiplate 384 plus, by PerkinElmer (1 mentions)
Versene, by Thermo Fisher Scientific (1 mentions)
Pherastar fs plate reader, by BMG Labtech (1 mentions)
Pherastar fs, by BMG Labtech (1 mentions)
Mithras lb 940, by Berthold Technologies (1 mentions)
6 protocols using htrf camp dynamic kit
1
cAMP Production Measurement Protocol
2
Teratoma Formation and cAMP Assay in iPSC Research
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The iPSCs cells (1 3 10 6 cells in 20-mL PBS) were injected into the testis of 8-to 10-week-old male CB17 SCID mice (Charles River). Teratomas were resected 9-12 weeks after injection, fixed with 10% neutralbuffered formalin for 24 h, and paraffin-embedded sections (4 mm) were processed and stained with hematoxylin and eosin. All mouse studies were carried out according to protocols approved by the Animal Research Committee of Tokyo Dental College (No. 270401).
cAMP assay cAMP accumulation was measured using homogeneous time-resolved fluorescence (HTRF) cAMP dynamic kits (CisBio, Codolet, France) according to the manufacturer's protocol. iPSCs were cultured in 96-well plates (20,000 cells/well) and treated with parathyroid hormone (PTH) (100 nM; Sigma-Aldrich, Buchs, Switzerland) 24 or adrenocorticotropic hormone (ACTH) (10 nM; Sigma-Aldrich, Buchs, Switzerland) 25 along with 500-nM 3-isobutyl-1-methylxanthine (IBMX; Cayman Chemical, Ann Arbor, MI, USA). At each time point, the cells were lysed with 1% Triton X-100. cAMP accumulation was detected by HTRF (l ex = 330 nm, l em = 665 and 620 nm) using a microplate reader (Synergy, BioTek, Winooski, VT, USA).
cAMP assay cAMP accumulation was measured using homogeneous time-resolved fluorescence (HTRF) cAMP dynamic kits (CisBio, Codolet, France) according to the manufacturer's protocol. iPSCs were cultured in 96-well plates (20,000 cells/well) and treated with parathyroid hormone (PTH) (100 nM; Sigma-Aldrich, Buchs, Switzerland) 24 or adrenocorticotropic hormone (ACTH) (10 nM; Sigma-Aldrich, Buchs, Switzerland) 25 along with 500-nM 3-isobutyl-1-methylxanthine (IBMX; Cayman Chemical, Ann Arbor, MI, USA). At each time point, the cells were lysed with 1% Triton X-100. cAMP accumulation was detected by HTRF (l ex = 330 nm, l em = 665 and 620 nm) using a microplate reader (Synergy, BioTek, Winooski, VT, USA).
3
Quantifying cAMP and IP1 Dynamics
4
Secretin-Induced cAMP Assay Protocol
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cAMP assays were performed using a homogeneous time-resolved fluorescence (HTRF®) cAMP dynamic kit (CisBio Bioassays, Codolet, France). Cells were detached by incubation at 37°C for 5–10 min with Versene (Invitrogen, Paisley, U.K.), counted and added at 5000 cells/well to low-volume 384-well plates (Proxi-plate™ 384 Plus, PerkinElmer Life Sciences). cAMP production was stimulated by the addition of varying concentrations of secretin followed by a 30 min incubation at room temperature. Outputs were measured by using a PHERAstar FS plate reader (BMG Labtech Ltd, Buckinghamshire, U.K.).
5
Forskolin-Stimulated cAMP Production Assay
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cAMP assays were performed using a homogeneous time-resolved fluorescence (HTRF®) cAMP dynamic kit (CisBio Bioassays; CisBio, Codolet, France). Cells were plated at 2000 cells/well in low-volume 384-well plates, and inhibition of 1 μm forskolin-stimulated cAMP production was assessed following a 30-min co-incubation with test compounds. Outputs were measured by using a PHERAstar FS plate reader (BMG Labtech, Aylesbury, UK).
6
Quantification of Agonist-Induced cAMP
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Quantification of agonist-induced rise of intracellular cAMP was performed using CHO-hM2 cells pretreated with 100 ng/ml -1 PTX for 16-22 h, as described previously using a HTRF-cAMP dynamic kit (Cisbio, Bagnols-sur-Cèze, France) following the manufacturer´s instructions. cAMP content was detected dispensing 50,000 cells per well in a buffer containing Hanks´ balanced salt solution with 20 mM HEPES and 1 mM of the phosphodiesterase inhibitor IBMX. Fluorescence was quantified on a Mithras LB 940 multimode reader (Berthold Technologies, Bad Wildbad, Germany). Levels of cAMP were normalized to the amount of cAMP generated by 100 µM Acetylcholine (ACh).
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