Bridge-It® Cyclic AMP (cAMP) Designer Assay Kit, 96-well format

cAMP 3

Clear selection

Description

The Bridge-It® Cyclic AMP (cAMP) assay is highly specific. ATP, AMP, and cGMP have all been tested for selectivity using the cAMP assay. No response was detected using the Bridge-It® cAMP assay with any of these compounds within the concentration range that would be expected to occur in “real life” samples (i.e., millimolar range for AMP and ATP, and micromolar range for cGMP). The sensitivity and performance characteristics of the Bridge-It®cAMP designer and the Bridge-It® all in one assay products are presented in the following Table and Figures.

Bridge-It® cAMP Assay

Microplate Format

cAMP Detection Level

designer

96-well

5nM (0.5 pmol/well in 100 µl vol.)

all in one

384-well

5nM (100 fmol/well in 20 µl vol)

Pricing: The 50-well kit is a one-time trial purchase. For bulk pricing options, please contact us. For any other questions or comments, please do not hesitate to contact us. We are always happy to help!

 

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Example of Bridge-It® cAMP designer Assay for Attached Cells

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The Bridge-It® cAMP designer assay allows for the stimulation and measurement of cAMP in adherent monolayer cells attached to the wells of a tissue culture microplate. In the following example, HEK 293 cells were trypsinized and plated into the wells of a 96-well polystyrene tissue culture microplate at a density of 25,000 cells per well in 50 µl of culture media. The cells were allowed to attach to the bottom of the wells overnight. The media was removed the next day and the wells were washed with a buffered saline solution. The wash buffer was then removed and replaced with 50 µl of KRB-IBMX buffer. For stimulation, 1 µl of forskolin at different concentrations was added to each well and incubated and rotated gently for 15 minutes at ~25ºC. To quantify cAMP in each well, the forskolin buffer solution was removed, 100 µl of the designer Assay Solution was added to each well, and the microplate was incubated for 30 minutes at ~25ºC with rotation. The well contents were then transferred into a black 96-well microplate and the fluorescent signal was read using the settings for fluorescein (~480 nm excitation, ~520 nm emission).

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Cell Preparation and Forskolin Stimulation – HEK-293 cells were grown using standard cell culture conditions to ~70-80% confluency. The cells were trypsinized, harvested, and washed in serum-free Krebs Ringers Bicarbonate buffer containing a phosphodiesterase inhibitor (i.e., KRB-IBMX buffer). Cyclic AMP (cAMP) levels were measured following forskolin stimulation of the cells using the Bridge-It® designer assay. Following incubation with Bridge-It® Assay Solution for 30 min at ~ 25°C, the fluorescent signal was read at ~480 nm excitation, ~520 nm emission. Data analysis was performed using relative fluorescence (RF) as a signal change relative to a blank (RF= (F0-F)/F0). RF analysis has been shown to be highly reproducible and does not depend greatly on the instrument used to read fluorescence. Examples can be seen below.

Example of Bridge-It® cAMP designer Assay for Cells in Suspension

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For information on the assay principle please see the Bridge-It Assay Technology platform page or download the protocol.

This product is protected by patents and pending patents.


 

Selected Customer Publications using Mediomics’ Bridge-It cAMP designer Assay Kit:

1. Oh SK, et al. Pannexin 3 is required for normal progression of skeletal development in vertebrates. The FASEB Journal. 2015 July 16.

— “Measurement of intracellular cAMP After 4 d of micromass culture, the cAMP levels in the cells were measured using the Bridge-It cAMP Designer fluorescence assay kit (Mediomics, St. Louis, MO, USA).”

2. Salvatori AS, Elrick MM, Samson WK, Corbett JA, Yosten GL. Neuronostatin inhibits glucose-stimulated insulin secretion via direct action on the pancreatic α-cell. Am J Physiol Endocrinol Metab. 2014 Jun 1;306(11):E1257-63.

–“Sample pellets were resuspended in appropriate assay buffers, and cAMP was measured by radioimmunoassay (PerkinElmer), BioTrak enzyme immunoassay (Amersham, Piscataway, NJ), or Bridge-It fluorometric assay (Mediomics, St. Louis, MO) according to the manufacturers’ protocols.”

3. Saalau-Bethell SM, Berdini V, Cleasby A, Congreve M, Coyle JE, Lock V, Murray CW, O’Brien MA, Rich SJ, Sambrook T, Vinkovic M, Yon JR, Jhoti H. Crystal structure of human soluble adenylate cyclase reveals a distinct, highly flexible allosteric bicarbonate binding pocket.ChemMedChem. 2014 Apr;9(4):823-32.

–“The enzymatic activity of hsolAC was measured in 96-well (black) plate format using the Mediomics Bridge-It designer cAMP kit (cat #122 935, Mediomics, LLC, St. Louis, MO, USA).”

4. Ishikawa M1, Iwamoto T, Fukumoto S, Yamada Y. Pannexin 3 inhibits proliferation of osteoprogenitor cells by regulating Wnt and p21 signaling. J Biol Chem. 2014 Jan 31;289(5):2839-51.

–“The level of cAMP was determined with a Bridge-It cAMP designer fluorescence assay kit (Mediomics).”

5. Mandal S, Mukhopadhyay S, Bandhopadhyay S, Sen G, Biswas T. 14-Deoxyandrographolide alleviates ethanol-induced hepatosteatosis through stimulation of AMP-activated protein kinase activity in rats. Alcohol. 2014 Mar;48(2):123-32

–“Intracellular cAMP levels in rat hepatocytes and human hepatoma HepG2 cells (10 5 /mL) were measured by using Bridge-It ® cAMP Fluorescence Assay kits (Mediomics, LLC).”

6. Masyuk AI, et al. Ciliary subcellular localization of TGR5 determines the cholangiocyte functional response to bile acid signaling. Am J Physiol Gastrointest Liver Physiol. 2013 Jun 1;304(11):G1013-24.

–“Measurement of cAMP. cAMP levels in cholangiocytes were measured by the Bridge-It cAMP designer cAMP assay (Mediomics).”

7. Lee YS et al. The fractalkine/CX3CR1 system regulates β cell function and insulin secretion. Cell. 2013 Apr 11;153(2):413-25.

–“Intracellular cAMP level of Min6 cells was measured using Bridge-it cAMP assay kit (Mediomics, USA).”

8. Nan YS, et al. Different effects of dibutyryl cAMP on monolayer permeability in human aortic and coronary arterial endothelial cells.African Journal of Microbiology Research Vol.6(5) 897-903, 2012

–“Measurement of intracellular cAMP in endothelial cells Intracellular cAMP was measured using a Bridge-It cAMP designer fluorescence assay kit (Mediomics, St. Louis, MO, USA) according to the manufacturer’s instructions.”

9. Kanellopoulos AK, Semelidou O, Kotini AG, Anezaki M, Skoulakis EM. Learning and memory deficits consequent to reduction of the fragile X mental retardation protein result from metabotropic glutamate receptor-mediated inhibition of cAMP signaling in Drosophila. J Neurosci. 2012 Sep 19;32(38):13111-24.

–“The Bridge-It cAMP designer fluorescence assay system (Mediomics) was used to measure cAMP levels in all samples.”

10. McGraw, DW. A Polymorphic Change in Amino Acid 220 (Ser220Cys) Of The Human ß2- Adrenergic Receptor Encodes Receptors With Different Gs-Coupled Signal Transduction Properties.

–“Cellular production of 125 cAMP was measured using a fluorescent assay kit (Mediomics, MO).”

11. Abukhashim M, Wiebe GJ, Seubert JM. Regulation of forskolin-induced cAMP production by cytochrome P450 epoxygenase metabolites of arachidonic acid in HEK293 cells. Cell Biol Toxicol. 2011 Oct;27(5):321-32.

–“Intracellular cAMP levels were measured by using commercially available kits (Mediomics).”

12. Reszka KJ, Sallans L, Macha S, Brown K, McGraw DW, Kovacic MB, Britigan BE. Airway peroxidases catalyze nitration of the {beta}2-agonist salbutamol and decrease its pharmacological activity. J Pharmacol Exp Ther. 2011 Feb;336(2):440-9.

–“The ability of native and nitrated salbutamol to stimulate cAMP production in airway smooth muscle cells was measured by a fluorescent detection assay using a commercially available kit (Mediomics, St. Louis, MO).”

13. Iwamoto T, Nakamura T, Doyle A, Ishikawa M, de Vega S, Fukumoto S, Yamada Y. Pannexin 3 regulates intracellular ATP/cAMP levels and promotes chondrocyte differentiation. J Biol Chem. 2010 Jun 11;285(24):18948-58.

–“The level of cAMP was determined with a Bridge-It cAMP Designer fluorescence assay kit (Mediomics).”

14. Nifoussi SK. Posttranslational regulation of protein function and stability at the mitochondria and beyond. PhD (Doctor of Philosophy) thesis, University of Iowa, 2010.

–“…Bridge-It cAMP designer assay (Mediomics) was used based on manufacturers protocol.”

15. Furukawa F, Kanehara S, Harano F, Shinohara S, Kamimura J, Kawabata S, Igarashi S, Kawamura M, Yamamoto Y, Miyachi Y. Effects of adenosine 5′-monophosphate on epidermal turnover. Arch Dermatol Res. 2008 Oct;300(9):485-93.

–“Intracellular cAMP level was measured with Bridge-It cAMP designer fluorescence assay kit (Mediomics, LLC, St. Louis, MO, USA).”

16. Anatoliy I. Masyuk, Sergio A. Gradilone, Jesus M. Banales, Bing Q. Huang, Tatyana V. Masyuk, Seung-Ok Lee, Patrick L. Splinter, Angela J. Stroope, and Nicholas F. LaRusso. Cholangiocyte primary cilia are chemosensory organelles that detect biliary nucleotides via P2Y12 purinergic receptors. Am J Physiol Gastrointest Liver Physiol. 2008 October; 295(4): G725–G734.

–“cAMP levels were measured by the Bridge-It cAMP designer cAMP assay (Mediomics) and expressed in pmol of cAMP per well.”

17. Masyuk TV, Masyuk AI, Torres VE, Harris PC, Larusso NF. Octreotide inhibits hepatic cystogenesis in a rodent model of polycystic liver disease by reducing cholangiocyte adenosine 3′,5′-cyclic monophosphate. Gastroenterology. 2007 Mar;132(3):1104-16.

–“cAMP concentrations were determined by the Bridge-It cAMP designer cAMP assay (Mediomics, LLC, St Louis, MO).”

18. Daniel J. Kelley, Richard J. Davidson, Jamie L. Elliott, Garet P. Lahvis, Jerry C. P. Yin, and Anita Bhattacharyya.The Cyclic AMP Cascade Is Altered in the Fragile X Nervous System. PLoS ONE. 2007; 2(9): e931.

–“The Bridge-It cAMP designer fluorescence assay system (Mediomics, LLC, St. Louis, MO) was used to measure cyclic AMP levels in all samples.”

19. Anatoliy I. Masyuk, Tatyana V. Masyuk, Patrick L. Splinter, Bing Q. Huang, Angela J. Stroope, and Nicholas F. LaRusso (2007)Cholangiocyte Cilia Detect Changes in Luminal Fluid Flow and Transmit Them into Intracellular Ca2+ and cAMP Signaling. Gastroenterology. 2006 September; 131(3): 911–920.

–“The concentrations of cAMP were determined by the Bridge-It™ cAMP designer cAMP assay (Mediomics, LLC, St. Louis, MO).”

20. Brent D. Wilson, Masaaki Ii, Kye Won Park, Arminda Suli, Gerhardus A.H. Kock, Lise K. Sorensen, Wonhee Suh, Fréderic Larrieu-Lahargue, Lisa D. Urness, Kirk R. Thomas, Chi-Bin Chien, Douglas W. Losordo, and Dean Y. Li.Netrins Promote Developmental and Therapeutic Angiogenesis. Science. 2006 August 4; 313(5787): 640–644.

–“Two different cAMP measurement systems were utilized (Mediomics and Assay Designs); similar results were obtained with each assessment.”

21. Kalhan R, Thavarajah K, Nlend MC, Nair A, Smith LJ, Sporn PHS. The soy isoflavone genistein blocks transforming growth factor b1‐stimulated lung fibroblast to myofibroblast transformation. Journal of Investigative Medicine: 1 March 2006 – Volume 54 – Issue 2 – p S354.

–“Using an assay in which reductions in fluores- cence intensity are associated with increasing cAMP levels (Mediomics, LLC), we quantified cAMP levels in neurospheres (NS) and differentiated cells (DC) from the human cortical fragile X (M049) and control…”

22. Brennan JP, Bardswell SC, Burgoyne JR, Fuller W, Schroder E, Wait R, Begum S, Kentish JC, Eaton P. (2006)Oxidant-induced Activation of Type I Protein Kinase A Is Mediated by RI Subunit Interprotein Disulfide Bond Formation. J. Biol. Chem. 281: 21827-21836.

–“cAMP was measured in cells treated with H2O2 or isoprenaline using a Bridge-It cAMP designer fluorescence assay kit according to the manufacturer’s guidelines (Mediomics).”

23. Zhang X, Candas M, Griko NB, Taussig R, Bulla LA Jr. (2006) A mechanism of cell death involving an adenylyl cyclase/PKA signaling pathway is induced by the Cry1Ab toxin of Bacillus thuringiensisProc Natl Acad Sci U S A.103:9897-9902.

–“Involvement of AC/PKA pathway in Cry1Ab toxin-induced cytotoxicity. (A) Stimulation of cAMP production by Cry1Ab toxin. Cells were incubated with isobutyl-1-methylxanthine (0.5 mM) before the addition of Cry1Ab, and cAMP was measured by using the Bridge-It cAMP Assay (Mediomics) in S5 and H5 cells (3 x 104).”

24. White CA, Sowa JR, Blake AD. A fluorescence-based second messenger assay to identify small molecule antagonists of somatostatin. 2006, ASPET, 48 (1).

–“Intracellular cAMP levels were quantified using a Bridge-It cAMP Designer Fluorescence Assay (Mediomics, LLC, St Louis, MO), which replaced more conventional radioimmunoassay methods.”

25. Keys JR, Zhou RH, Harris DM, Druckman CA, Eckhart AD. (2005) Vascular smooth muscle overexpression of G protein-coupled receptor kinase 5 elevates blood pressure, which segregates with sex and is dependent on Gi-mediated signalingCirculation. 112:1145-1153.

–“cAMP accumulation was measured by use of a Bridge-It cAMP assay kit according to the manufacturer’s instructions (Mediomics, LLC).”

26. Calebiro D, de Filippis T, Lucchi S, Covino C, Panigone S, Beck-Peccoz P, Dunlap D, Persani L. (2005) Intracellular entrapment of wild-type TSH receptor by oligomerization with mutants linked to dominant TSH resistanceHum Mol Genet.14:2991-3002.

–“cAMP assay was performed using Bridge-It cAMP Designer Fluorescence Assay (Mediomics LLC) in 96-well, black, round-bottom plates, according to manufacturer’s instructions.”


This product is protected by patents and pending patents.

Catalog #: 122934 (50 measurements) , 122935 (96 measurements)

Protocol: 

SDS: