The determination of intracellular cAMP concentrations was modified from the previously described protocol (Zhang et al., 2017 (link); Zou et al., 2019 (link)). After 24 h of transfection, all the wells were added with 10 μM ADP (Sigma, United States) and incubated at 37°C for 5 min (Zhu et al., 2015 (link)). The mixture was collected in 100 μl phosphate-buffered saline, frozen at −80°C for 45 min, and then thawed in a 37°C water bath three times. After being centrifuged at 1,500 × g for 10 min, the supernatant was collected. Also, the cAMP Elisa Kit (Elabscience Biotechnology, China) was used to quantify cAMP concentrations.
Camp elisa kit
Manufactured by Elabscience
Sourced in China, United States
The CAMP ELISA Kit is a laboratory assay designed for the quantitative detection of cyclic adenosine monophosphate (cAMP) levels in various sample types. The kit utilizes the enzyme-linked immunosorbent assay (ELISA) principle to measure cAMP concentrations.
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Lab products found in correlation
Adenosine diphosphate (adp), by Merck Group (1 mentions)
Calcineurin assay kit, by Nanjing Jiancheng (1 mentions)
Succinate dehydrogenase assay kit, by Nanjing Jiancheng (1 mentions)
Succinate colorimetric assay kit, by Merck Group (1 mentions)
Mouse il 1β, by Cusabio (1 mentions)
Il 6 elisa kit, by Cusabio (1 mentions)
Il 1β, by Cusabio (1 mentions)
Pka colorimetric activity kit, by Thermo Fisher Scientific (1 mentions)
Ab32096, by Abcam (1 mentions)
Ab171750, by Abcam (1 mentions)
Ab75991, by Abcam (1 mentions)
Rolipram, by MedChemExpress (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Nitric oxide assay kit, by Beyotime (1 mentions)
12 protocols using camp elisa kit
1
Intracellular cAMP Quantification Protocol
2
Immunological and Metabolic Markers Quantification
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The blood samples collected from the experimental animal were allowed to stand and centrifuged at 1000× g for 15 min to obtain serum. The mouse IL-1β and IL-6 ELISA Kit (CUSABIO, Wuhan, China) were used to determine the levels of IL-1β and IL-6, respectively.
After treating bEnd.3 cells with the corresponding drugs, the supernatant was collected for the measurement of IL-1β (CUSABIO, Wuhan, China). In addition, the treated bEnd.3 cells were washed with PBS and collected with cell lysis buffer. After centrifuging at 12,000× g for 15 min at 4 °C, the supernatant was collected for the measurement of sAC, cAMP, and CaMKII according to the protocol of sAC ELISA Kit, cAMP ELISA Kit (Elabscience, Wuhan, China) and CaMKII ELISA Kit (Meimian, Yancheng, China). The quantification of succinate was measured by succinate colorimetric assay kit (Sigma, MAK184, St. Louis, MO, USA) The activity of CaN and SDH were detected according to the calcineurin assay kit (Jiancheng, A068-1-1, Nanjing, China) and succinate dehydrogenase assay kit (Jiancheng, A022-1-1, Nanjing, China).
After treating bEnd.3 cells with the corresponding drugs, the supernatant was collected for the measurement of IL-1β (CUSABIO, Wuhan, China). In addition, the treated bEnd.3 cells were washed with PBS and collected with cell lysis buffer. After centrifuging at 12,000× g for 15 min at 4 °C, the supernatant was collected for the measurement of sAC, cAMP, and CaMKII according to the protocol of sAC ELISA Kit, cAMP ELISA Kit (Elabscience, Wuhan, China) and CaMKII ELISA Kit (Meimian, Yancheng, China). The quantification of succinate was measured by succinate colorimetric assay kit (Sigma, MAK184, St. Louis, MO, USA) The activity of CaN and SDH were detected according to the calcineurin assay kit (Jiancheng, A068-1-1, Nanjing, China) and succinate dehydrogenase assay kit (Jiancheng, A022-1-1, Nanjing, China).
3
cAMP Quantification in MIN6 Cells
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To measure cAMP levels in MIN6 cells, phosphate‐buffered saline (PBS) was added to MIN6 cells and repeatedly freeze‐thawed, centrifuged at 10 000 g for 10 min, and the particles were removed. MIN6 cells were repeatedly freeze‐thawed in PBS and centrifuged 10 000 g for 10 min to remove particles. The supernatants were collected and the cAMP content was determined using cAMP ELISA Kit (Elabscience, China) according to the manufacturer's instructions.
4
Quantifying Cytokine and Kinase Response
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Cells (1 × 106) with complete growth medium were seeded in 6-well plates and cultured overnight. On the following day, cells were washed with PBS and treated with 5 ng/ml IFNγ for 24 hours. The concentrations of mouse CXCL9/MIG, human cAMP level and the activity of PKA in the supernatants were analyzed using CXCL9 ELISA kit (R&D Systems, MCX900), cAMP ELISA Kit (Elabscience, E-EL-0056c) and PKA Colorimetric Activity Kit (Thermo Fisher scientific, EIAPKA) respectively, according to the manufacturer’s protocols. Microplate reader was set to 450 nm to determine the optical density.
5
Content of cyclic adenosine monophosphate in NK cells was detected by enzyme-linked immunosorbent assay
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Splenic NK cells (3 × 105) (cell viability: 95.1–96.7%) were collected after co-incubation and resuspended with 300 µL PBS containing 10 µM 3-Isobutyl-1-methylxanthine (Beyotime Biotechnology). Cells were broken using an ultrasound crusher and were centrifuged at 3000 g for 15 min at 4 °C. The supernatants were measured for cyclic adenosine monophosphate (cAMP) levels using the cAMP ELISA Kit (Elabscience, Bethesda, USA) according to the manufacturer’s instructions.
6
Intracellular cAMP Concentration Assay
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The effect of TS on the intracellular cAMP concentrations was measured as previously described with some modifications (21 (link)). YEM30 cells (1×106 cells/mL) with various doses of TS were cultured in RPMI1640 medium at 37 °C for 12 h, and 2 µg/mL FLC served as positive control group. Cells were harvested by centrifugation, washed 3 times with deionized water, weighed the dry weight, frozen in liquid nitrogen and thawed at room temperature repeatedly, and finally suspended in deionized water with 5% trichloroacetic acid. Following breaking by ultrasonication, the supernatant was neutralized with water-saturated ether five times and then subjected to freeze-drying. The content of intracellular cAMP was measured using a cAMP ELISA Kit (Elabscience Biotechnology Co., Ltd, Guangdong, China) according to the provided protocol. The concentrations of cAMP were modified by their cell weights.
7
Quantification of Cellular cAMP Levels
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Cellular cAMP assay was performed as previously described [19 (link)]. In brief, cultured primary hepatocytes (5 × 106) were collected and resuspended with 200 μL PBS, then hepatocytes were broken using an ultrasound crusher and were centrifuged at 3000g for 15 min at 4 °C. For hepatic tissue, the samples were accurately weighted, added with 10X volume of PBS, homogenized on ice using a Tissue Homogenizer, and then centrifuged at 3000g for 15 min at 4 °C. The cellular and hepatic tissue supernatants were measured for cyclic adenosine monophosphate (cAMP) levels using the cAMP ELISA Kit (Elabscience, Bethesda, USA) according to the manufacturer's instructions.
8
Intracellular cAMP Concentration Measurement
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The intracellular cAMP concentration was measured according to the previous method with some modifications [15 (link)]. YEM30 strain treated with different doses of MPD was incubated in RPMI 1640 medium at 37°C. After 12 h incubation, the intracellular cAMP concentration was measured by a cAMP ELISA Kit (Elabscience Biotechnology Co., China).
9
Rolipram and Nitric Oxide Regulation
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Rolipram was purchased from Medchem Express. Tetrodotoxin (TTX), Nω‐Nitro‐L‐arginine (L‐NNA), Rp‐Adenosine 3′,5′‐cyclic monophosphorothioate triethylammonium salt hydrate (Rp‐cAMPS) were all purchased from Sigma‐Aldrich. Primary antibodies were used as follows: the rabbit anti‐PDE4D(ab171750, Abcam), the rabbit anti‐p‐CREB (ab32096, Abcam), and the rabbit anti‐PKA(ab75991, Abcam), the rabbit anti‐nNOS (4231, Cell signaling), BCA protein assay kit (Beyotime).
The Nitric Oxide Assay kit was bought from Beyotime (S0021S, Beyotime), and cAMP ELISA kit was from Elabscience(E‐EL‐0056c, Elabscience).
TTX, L‐NNA, and Rp‐cAMPS were dissolved in Tyrode's buffer. In contraction recordings of colonic muscle strips in vitro, the dimethylsulfoxide (DMSO) concentration was <0.01%, and the rest of the diluent for Rolipram was Tyrode's buffer. In experiments of intraperitoneal administration in rats, the diluent for Rolipram was DMSO (the added concentration of DMSO was 10%), 70% PEG300, 10% Tween‐80, and 10% saline, so the final DMSO concentration was 1%.
The Nitric Oxide Assay kit was bought from Beyotime (S0021S, Beyotime), and cAMP ELISA kit was from Elabscience(E‐EL‐0056c, Elabscience).
TTX, L‐NNA, and Rp‐cAMPS were dissolved in Tyrode's buffer. In contraction recordings of colonic muscle strips in vitro, the dimethylsulfoxide (DMSO) concentration was <0.01%, and the rest of the diluent for Rolipram was Tyrode's buffer. In experiments of intraperitoneal administration in rats, the diluent for Rolipram was DMSO (the added concentration of DMSO was 10%), 70% PEG300, 10% Tween‐80, and 10% saline, so the final DMSO concentration was 1%.
10
Measuring Astrocyte cAMP Levels
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To measure cyclic adenosine monophosphate (cAMP) level in astrocyte, cAMP concentration was detected using cAMP ELISA kit (Elabscience, USA) according to the manufacturer’s instructions. Astrocyte cell lysate was mixed with biotinylated detection Ab solution and incubated for 45 min at 37 ℃. And then, after incubation, the HRP conjugate solution was added, followed by 30 min incubation at 37 ℃. After incubation, substrate reagent was added and incubated for 15 min at 37 ℃ in the dark. Finally, after incubation, stop solution was added and cAMP concentration was detected at 450 nm using an Epoch microplate reader.
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