Intracellular cAMP concentration ([cAMP]i) was determined as previously described (Huang et al., 2007 (link)). Cells were plated into 12-well tissue culture plates (Nunc, Roskilde, Denmark) at a density of 1× 106 cells per well and incubated at 37°C with 10% CO2 in a humidified incubator. The cells were pre-incubated in Dulbecco’s phosphate-buffered saline (DPBS; Gibco-Invitrogen) containing 100 uM phosphodiesterase inhibitor IBMX for 20 min at room temperature. After the preincubation, a 50-μl aliquot of D-PBS containing various concentrations of reagents was added. The culture was then incubated for 20 min at room temperature. The reaction was stopped by aspirating the solution and then adding 250 μl ice-cold cell lysis buffer immediately to lysis the cells. The cell lysate was scraped into 1.5 ml Eppendorf tubes for collections and stored at –70°C until use. The solution was centrifugated and [cAMP]i in the supernatant was determined using a cAMP ELISA kit (R&D Systems, Minneapolis, MN).
Camp elisa kit
Manufactured by R&D Systems
Sourced in United States
The CAMP ELISA kit is a laboratory assay used to quantify levels of the cyclic AMP response element-binding protein (CREB) in biological samples. It utilizes an enzyme-linked immunosorbent assay (ELISA) format to detect and measure the target protein. The kit provides the necessary reagents and protocols to perform this analysis.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
12 well tissue culture plate, by Thermo Fisher Scientific (1 mentions)
Dulbecco s phosphate buffered saline dpbs, by Thermo Fisher Scientific (1 mentions)
Isobutyryl methylxanthine, by Merck Group (1 mentions)
Dmem f 12 glutamax, by Thermo Fisher Scientific (1 mentions)
γ tubulina, by Merck Group (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Bcl 2, by Santa Cruz Biotechnology (1 mentions)
Cell proliferation kit 2 xtt, by Roche (1 mentions)
Nci h2452, by American Type Culture Collection (1 mentions)
Msto 211h, by American Type Culture Collection (1 mentions)
Pemetrexed, by Eli Lilly (1 mentions)
V cholerae neuraminidase, by Roche (1 mentions)
T84 cells, by American Type Culture Collection (1 mentions)
H4784, by Merck Group (1 mentions)
Cea908ge, by USCN (1 mentions)
Cell incubator, by Thermo Fisher Scientific (1 mentions)
Synergy 2 multifunctional microplate reader, by Agilent Technologies (1 mentions)
High speed refrigerated centrifuge, by Thermo Fisher Scientific (1 mentions)
Pluronic f 127, by Thermo Fisher Scientific (1 mentions)
13 protocols using camp elisa kit
1
Determining Intracellular cAMP Levels
2
Quantifying cAMP Levels in Cells
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Isobutyryl methylxanthine (Sigma‐Aldrich) was added to the cells to suppress phosphodiesterase. The cells were then homogenized in ice‐cold 1 mol/L TCA and centrifuged at 2500 g RCF to precipitate the cells. The cAMP ELISA kit (R&D Systems) was then used to determine the concentration of cAMP in the lysates.
3
Evaluating Mesothelioma Cell Line Responses to Exemestane Treatment
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The human pleural mesothelial cell Met-5A and the human pleural MPM cell lines MSTO-211H (MSTO) and NCIH-2452 (NCI) were obtained from the American Type Culture Collection (ATCC) (Rockville, Md) and Ist-Mes1, Ist-Mes2, and MPP89 were obtained from Genova Institute Culture Collection. Cell lines were cultured as described previously [38 (link)]. Before treatment with exemestane, all cell lines were gradually conditioned in DMEM/F12 + Glutamax (Invitrogen) supplemented with 10% FBS and antibiotics.
The cell proliferation kit II (XTT) was purchased from Roche Molecular Biochemicals, Indianapolis, cAMP ELISA Kit from R&D Systems, the siRNA CD44 (5′GAACGAAUCCUGAAGACAU 3′, as 5′AUGUCUUCAGGAUUCGUUC3′) from Sigma, lipofectamine 2000 from Invitrogen, exemestane from Sequoia Research, Pemetrexed (Alimta) from Eli Lilly & Co, Cisplatino from Pfizer and Vitamina B12 (Dobetin) from Angelini SPA. Commercially available antibodies were used for immunoblo detection of: Bcl-2, p21, pCreb and CD44 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bcl- ×L (Cell Signaling Technology) and γ-tubulina (Sigma, Saint Louis Missouri, USA).
The cell proliferation kit II (XTT) was purchased from Roche Molecular Biochemicals, Indianapolis, cAMP ELISA Kit from R&D Systems, the siRNA CD44 (5′GAACGAAUCCUGAAGACAU 3′, as 5′AUGUCUUCAGGAUUCGUUC3′) from Sigma, lipofectamine 2000 from Invitrogen, exemestane from Sequoia Research, Pemetrexed (Alimta) from Eli Lilly & Co, Cisplatino from Pfizer and Vitamina B12 (Dobetin) from Angelini SPA. Commercially available antibodies were used for immunoblo detection of: Bcl-2, p21, pCreb and CD44 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bcl- ×L (Cell Signaling Technology) and γ-tubulina (Sigma, Saint Louis Missouri, USA).
4
cAMP Levels in Cortical Neurons
5
Colorectal Carcinoma Cell Culture
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T84 cells (American Type Culture Collection—CCL-248), a line derived from a lung metastasis of a human colorectal carcinoma, were used as previously described [55 (link)]. The active (AB5) form of Ctx, 4 kDa labeled FITC-dextran and 1,2-diamino-4,5-methylenedioxybenzene (DMB) were obtained from Sigma-Aldrich (St. Louis, MO, USA). V. cholerae neuraminidase was purchased from Roche (Clovis, CA, USA). The cAMP ELISA kit was from R&D Systems (Minneapolis, MN, USA). V. cholerae strain N16961 was acquired from ATCC (Manassas, VA, USA).
6
Melatonin Signaling in Avian Cells
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All of the blood samples were heparinized with 1000 UI/mL of sodium heparin (H4784, Sigma, St. Louis, MO, USA) in avian saline. After centrifugation at 1000× g for 20 min, the plasma was decanted and stored at −80 °C. Concentrations of MEL in plasma were measured by an enzyme-linked immunosorbent assay kit for anti-MEL (CEA908Ge, Uscn Life Science, Inc., Wuhan, China).
The cells were incubated with 1 μM of prazosin for 30 min followed by 250 pg/mL of MEL for 24 h, and 3-isobutyl-1-methylxanthin (IBMX 100 μM, T1713, Topscience, TX, USA) was added to the medium to prevent cAMP degradation before the cells were incubated for a further 30 min. The cells were collected and lysed with lysis buffer from the cAMP ELISA Kit (R&D Systems, Minneapolis, MN, USA). Further treatment of the samples was performed according to the manufacturer’s instructions.
The cells were incubated with 1 μM of prazosin or 10 μM of PD98059 for 30 min, followed by 250 pg/mL of MEL for 24 h. The supernatant was collected for IGF-I protein detection by the anti-chicken IGF-I ELISA kit (SEA050Ga, Uscn Life Science, Inc., Wuhan, China), according to the manufacturer’s protocols.
The cells were incubated with 1 μM of prazosin for 30 min followed by 250 pg/mL of MEL for 24 h, and 3-isobutyl-1-methylxanthin (IBMX 100 μM, T1713, Topscience, TX, USA) was added to the medium to prevent cAMP degradation before the cells were incubated for a further 30 min. The cells were collected and lysed with lysis buffer from the cAMP ELISA Kit (R&D Systems, Minneapolis, MN, USA). Further treatment of the samples was performed according to the manufacturer’s instructions.
The cells were incubated with 1 μM of prazosin or 10 μM of PD98059 for 30 min, followed by 250 pg/mL of MEL for 24 h. The supernatant was collected for IGF-I protein detection by the anti-chicken IGF-I ELISA kit (SEA050Ga, Uscn Life Science, Inc., Wuhan, China), according to the manufacturer’s protocols.
7
Measuring Cervical cAMP Levels
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Cervical tissue cAMP concentration was measured using the cAMP ELISA Kit (R&D Systems, Minneapolis, MN, USA). The sample was weighed immediately after it was collected, homogenized in 10 volumes of ice-cold 5% trichloroacetic acid, and centrifuged at 12,000 rpm for 10 min. The supernatant was extracted with three volumes of water-saturated ether. After drying, the extracts were stored at −80°C until the cAMP assay. The tissue cAMP level was expressed in pmol/mg tissue.
8
Adenosine A2A Receptor Assay in HEK293 Cells
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HEK293 cells
stably transfected with adenosine A2A (hADORA2A) were purchased from Shanghai Saiye. Dulbecco’s modified
Eagle’s medium (DMEM)/high sugar medium (BI), penicillin–streptomycin
solution, pancreatin cell digest (Sigma, USA), fetal bovine serum
(Gibco, USA), dimethyl sulfoxide (DMSO) (Biosharp, China), a cAMP
Elisa kit (R & D, USA), NECA (Sigma, USA), and ADA (Sigma, USA)
were used.
A Synergy 2 multifunctional microplate reader (Bio-Tek,
USA), a cell incubator (Thermo, USA), and a high-speed refrigerated
centrifuge (Thermo, USA) were also employed.
stably transfected with adenosine A2A (hADORA2A) were purchased from Shanghai Saiye. Dulbecco’s modified
Eagle’s medium (DMEM)/high sugar medium (BI), penicillin–streptomycin
solution, pancreatin cell digest (Sigma, USA), fetal bovine serum
(Gibco, USA), dimethyl sulfoxide (DMSO) (Biosharp, China), a cAMP
Elisa kit (R & D, USA), NECA (Sigma, USA), and ADA (Sigma, USA)
were used.
A Synergy 2 multifunctional microplate reader (Bio-Tek,
USA), a cell incubator (Thermo, USA), and a high-speed refrigerated
centrifuge (Thermo, USA) were also employed.
9
Primary Cell Culture Reagents
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Dulbecco’s modified Eagle’s medium/Ham’s F12 (DMEM/F12) medium and fetal bovine serum were purchased from Invitrogen-Gibco (Grand Island, NY, USA). The Papain dissociation system was purchased from Worthington Biochemical Co. (Lakewood, NJ, USA). The cGMP ELISA kit was purchased from Cell Biolabs Inc. (San Diego, CA, USA) and cAMP ELISA kit was purchased from R&D systems (Minneapolis, MN, USA). Fura-2/AM and Pluronic F-127 were obtained from Molecular Probes (Eugene, OR, USA). Other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).
10
Cellular cAMP Measurement After PTHR1 Transfection
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At 48 hours after PTHR1 transfection, the cells were washed with assay buffer (135 mM NaCl, 6 mM KCl, 1 mM MgCl2, 2.8 mM glucose, 1.2 mM CaCl2, and 20 mM HEPES, pH 7.4) and incubated for 15 minutes at 37°C in the vehicle which was composed of the same buffer containing 0.1% heat inactivated BSA, 1 mM isobutylmethylxanthine (IBMX, Sigma-Aldrich Inc., St. Louis, MO, USA) or 10 nM PTH dissolved in the vehicle. Buffer was then aspirated quickly, plates were frozen immediately in liquid nitrogen, and subsequently, the frozen cells were thawed directly into 50 mM HCl. The cellular cAMP in the acid extracts was measured using a cAMP ELISA Kit (R&D Systems, MN, USA). Data were expressed as nanomoles per well.
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