Stably transfected cells were cultured in 12-well plates at a density of 5×105 cells/well and preincubated in stimulation buffer consisting of Dulbecco's modified Eagle's medium with 500 µM IBMX and 10 mM MgCl2, at 37°C for 20 min. Cells were then stimulated with buffer containing either orexin-A (0.1–1,000 nM), orexin-B (0.1–1,000 nM) for 10 min at 37°C. cAMP levels were determined using a Cyclic AMP assay kit (Cell Signaling Technology, Inc.) according to the manufacturer's instructions. Optical density at 450 nm was measured using a microplate reader.
Cyclic amp assay kit
Manufactured by Cell Signaling Technology
Sourced in United States
The Cyclic AMP Assay Kit is a lab equipment product designed to measure the levels of cyclic adenosine monophosphate (cAMP) in biological samples. cAMP is a crucial second messenger involved in various cellular signaling pathways. The kit provides a reliable and quantitative method to determine cAMP concentrations in a range of sample types, such as cell lysates, tissue extracts, or body fluids.
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6 protocols using cyclic amp assay kit
1
Orexin-induced cAMP response assay
2
Intracellular cAMP Production in αTC1.9 Cells
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Before the experiments, αTC1.9 cells were seeded in 6-well plates for 24 h. On the day after seeding, the cells were preincubated in KRB buffer with 10 mM glucose. After 30 min, the cells were incubated with a high glucose concentration (30 mM) in KRB buffer for 15 min. The cells were then lysed in 1× cell lysis buffer (Cell Signaling Technology, Beverly, MA, USA) containing 1 mM phenylmethylsulfonyl fluoride dissolved in isopropanol. The protein concentration of 80 μg was determined using the Bradford assay kit (Bio-Rad Laboratories, Hercules, CA, USA), and equal amounts of proteins were loaded. Intracellular cAMP production was assessed using a cyclic AMP assay kit (#4339, Cell Signaling Technology, Beverly, MA, USA) according to the manufacturer’s instructions.
3
Quantifying Intracellular cAMP Levels
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The Cyclic AMP Assay Kit (Cell Signaling Technology, Danvers, MA, USA) was used for the quantified measurement of intracellular cyclic cAMP level. The cells were lysed with lysis buffer and collected. The assay was completed following the manufacturer’s instructions. The absorbance was measured at 450 nm with a luminescence microplate reader. The cAMP standard curve was used to calculate the absolute amount of cAMP in the test samples.
4
Quantifying cAMP and Acetyl-CoA in KGC Cells
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cAMP was measured using the Cyclic AMP Assay Kit (4339; Cell Signaling Technology) in KGC cells were grown on laminin-coated plates as above. Acetyl-CoA in Extended Data Fig. 8k was measured using the PicoProbe Acetyl-CoA Fluorometric Assay Kit (K317-100; Biovision). All assays were done according to the manufacturers’ protocols.
5
Measuring Intracellular and Secreted cAMP
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Amount of cAMP was analyzed using Cyclic AMP Assay Kit (Cell Signaling Technology, Danvers, MA). Briefly, overnight-cultured cells were pretreated with PDE inhibitors, probenecid or other inhibitors for 1 h and then stimulated with forskolin for varying length of times. Both cells and media were collected for cAMP measurement. cAMPs in cells and media were considered as cellular and secreted cAMP respectively. To investigate the importance of MRPs or PDE4s, cells were transfected with control siRNA or siRNA pools for distinct MRP or PDE isotype for 4 days prior to the analysis of cAMP.
6
Quantifying cAMP and Acetyl-CoA in KGC Cells
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