Urinary bladder tissues were incubated with phosphodiesterase inhibitors (IBMX 5 µM, Sigma-Aldrich, St. Louis, MO, USA) for 30 min and treated with betulinic acid (5 µM) for another 1 h. Sample lysates were collected, and intracellular cAMP levels were measured using a cAMP Assay Kit (Abcam, Cambridge, MA, USA). Differences between treatment with betulinic acid or no treatment were indicated as the cAMP levels increased in each group.
Camp assay kit
Manufactured by Abcam
Sourced in United Kingdom, United States
The CAMP Assay Kit is a laboratory tool designed to measure the levels of cyclic adenosine monophosphate (cAMP) in biological samples. cAMP is an important signaling molecule involved in various cellular processes. The kit provides a quantitative assessment of cAMP concentrations using a competitive enzyme immunoassay technique.
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Lab products found in correlation
Microplate reader, by Agilent Technologies (3 mentions)
Il 12, by Thermo Fisher Scientific (1 mentions)
Tgf β1, by Merck Group (1 mentions)
Forskolin, by Merck Group (1 mentions)
Interleukin 6 (il 6), by R&D Systems (1 mentions)
Magnetic separation, by Miltenyi Biotec (1 mentions)
Anti cd3 clone 145 tc11, by BioXCell (1 mentions)
Anti ifn γ neutralizing antibodies, by BioXCell (1 mentions)
Tgf β1, by R&D Systems (1 mentions)
Il 23, by R&D Systems (1 mentions)
Infinite m200 plate reader, by Tecan (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Caspase 1, by Novus Biologicals (1 mentions)
Ab138880, by Abcam (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
2 7 dichlorofluorescein diacetate dcfh da, by Thermo Fisher Scientific (1 mentions)
L dopa, by Merck Group (1 mentions)
Xylenol orange, by Merck Group (1 mentions)
Mitotempo, by Merck Group (1 mentions)
18 protocols using camp assay kit
1
Betulinic Acid Increases cAMP in Bladder
2
Th Differentiation and cAMP Measurement
3
Measuring Intracellular cAMP in Fibroblasts
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The intracellular cAMP levels in cultured human dermal fibroblasts were measured using cAMP assay kit (Abcam, Cambridge, UK) following the manufacturer’s protocols. Briefly, fibroblast cells were treated with adenosine and cordycepin for 2 min and harvested. Absorbance at 450 nm was measured using microplate reader (BioTek, Winooski, VT, USA). Background wavelength correction was done at 540 nm.
4
Quantifying Intracellular cAMP Levels
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The levels of cAMP were detected in the cells by using a cAMP Assay Kit (ab234585, Abcam, Cambridge, UK) in accordance with the manufacturer's protocol. Briefly, we first plated the cells overnight in growth medium at 5000 cells/well in a 96-well plate, aspirated off the cell growth medium when cultures reached 90% confluence, added 100 μl/well of cell lysis buffer, and incubated them at room temperature for 10 minutes. Then, the lysates were centrifuged for 10 minutes at 13,000 rpm and the clear supernatant extract was analyzed for cAMP levels following the manufacturer's protocol. Finally, the intracellular levels of cAMP were determined by measuring the absorbance at wave length of 405 nm using a Tecan Infinite M200 plate reader (Crailsheim, Germany).
5
Measuring cAMP and GLP-1 Receptor in H9c2 Cells
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H9c2 cells (BCRC No. 60096) were cultured as previously described [22 (link)]. Briefly, the H9c2 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, pH 7.2; Gibco-BRL Life Technologies, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum. The cells were plated at 6000 cells/cm2 and allowed to proliferate in the medium. The medium was replaced on the second day. Then, the cells were used for the subsequent experiments because H9c2 cells show similar responses as the primary rat neonatal cardiomyocytes [22 (link)]. H9c2 cells were incubated with phosphodiesterase inhibitors (IBMX 5 µM) for 30 min, and then treated with myricetin at indicated concentration for another 1 h. Sample lysates were then collected and the intracellular cAMP levels were measured using cAMP Assay Kit (Abcam, Cambridge, MA, USA). Variations between treatment with myricetin or not were indicated as the increased cAMP in each. Additionally, changes in GLP-1 receptor expression were determined in these samples using the assay of mRNA levels in H9c2 cells as described below.
6
cAMP Quantification in Airway Cells
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One hundred thousand cells per well were seeded onto six-well plates. At about 90% confluency, HNEC, HTEC and HBEC3-KT cultures were incubated with 0.5 µg/ml CyaA, CyaA-AC– or TUC buffer for 1 h. Then, the cells were detached and lysed. cAMP in cell lysates was quantified by a direct competitive ELISA according to the manufacturer’s instructions (cAMP Assay Kit; #ab65355; Abcam, Cambridge, UK). The ELISA assay kit utilises a recombinant protein G–coated 96-well plate to anchor cAMP polyclonal Abs onto the plate efficiently. The amount of cAMP-HRP bound to the plate was determined by reading the colorimetric HRP activity at OD450 nm. The measured cAMP values were normalised to the total protein content using the DC™ protein assay (Bio-Rad, Feldkirchen, Germany). We performed four independent experiments, each in duplicate.
7
Quantifying Cytokine Secretion and cAMP in Microglial Cells
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Cell-secreted cytokines were measured from the cell culture supernatant using commercially available ELISA kits for mouse TNF-α, IL-1β/IL-1F2 (R&D Systems, Minneapolis, MN, USA), IL-18 (MBL, Naka-Ku, Nagoya Aichi, Japan), and Caspase-1 (Novus Biologicals) according to the manufacturer’s protocols. BV2 cells were cultured in the presence of Aβ (2 µM), TDCA (400 ng/ml), or KH7 (4 μM) for 24 h. Cells were harvested and lysed with 0.1 M HCl to measure intracellular cAMP using a cAMP assay kit (Abcam) according to the manufacturer’s protocol.
8
Quantifying A2AR-Mediated cAMP Modulation
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In brief, A375 cells (3×106) were incubated for 30 min at 37 °C with either the A2AR agonist CGS21680 alone or CGS21680 in different concentrations together with 5 μM of caffeine. Cells after treatment were harvested and centrifuged at 1000 rpm for 5 min, lysed and cAMP content was quantified using a cAMP assay kit (Abcam, Cambridge, UK) according to the manufacturer's instructions.
9
Quantifying Intracellular cAMP Levels
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LβT2 cells and primary rat adenohypophysis cells were transfected with the FOXP2 overexpression plasmid or vector. The intracellular cAMP level was detected by a cAMP Assay Kit (ab65355, Abcam, UK) according to the manufacturer’s protocol. The amount of cAMP was determined by measuring the absorbance at OD 450 nm, and the level of signal was compared to a standard curve for free cAMP to obtain the intracellular cAMP level.
10
Intracellular cAMP Quantification Assay
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The cell lines were plated into 24 well-plates with an initial cell density of 2 × 105 cells/well, and treated with BAs at 25 μM for 1 h. The intracellular cAMP was extracted, and determined using a cAMP Assay Kit according to the manufacturer's instruction (ab138880, Abcam).
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