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Pde activity assay kit

Manufactured by Abcam
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The PDE Activity Assay Kit is a laboratory equipment designed to measure the enzymatic activity of phosphodiesterases (PDEs), a class of enzymes that play a crucial role in the regulation of cellular signaling pathways. The kit provides a simple and reliable method for quantifying PDE activity in a variety of biological samples, such as cell lysates, tissue homogenates, or purified enzyme preparations.

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Activity assay kits (2 citations)

12 protocols using pde activity assay kit

1

Adipocyte Mitochondrial Activity Assay

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Differentiated adipocytes were stimulated with PA or dimethyl succinate at given concentrations for 8 h. Cells were collected by centrifugation. After washing with cold PBS, resuspend cells in PBS were subjected to ultrasonication for four times, and then centrifuged to remove cellular debris. The protein concentration was determined by Bicinchoninic Acid Protein Assay kit. The activity was measured by succinate dehydrogenase assay kit (Jiancheng, Nanjing, China) or PDE activity assay kit (Abcam, Cambridge, USA).
Xiao N., Yang L.L., Yang Y.L., Liu L.W., Li J., Liu B., Liu K., Qi L.W, & Li P. (2017). Ginsenoside Rg5 Inhibits Succinate-Associated Lipolysis in Adipose Tissue and Prevents Muscle Insulin Resistance. Frontiers in Pharmacology, 8, 43.
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2

PDE4 Activity Assay Protocol

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Experiments were carried out following the instructions provided by the “PDE Activity Assay Kit” (ab139460, Abcam, Cambdrige, UK) using the modified method detailed by Araiz et al [63 (link)]. Stock solutions of the compounds were prepared with DMSO (Less than 1%). Recombinant human PDE4 was incubated with 0.5 μM 5′-cGMP and 10 μL 5′-nucleotidase under 30°C for 90 minutes. The kit based on the sequential hydrolysis of cyclic nucleotides by PDE4 and 5′-nucleotidase. The released phosphate by enzymatic cleavage was directly proportional to PDE4 activity. The reaction was terminated by incubation with termination buffer under room temperature for 20 minutes. OD 620nm were measured on a plate reader to determine the amount of GMP generated. For measurement of IC50, 6 concentrations of inhibitors were used under the substrate concentration the suitable enzyme concentration. The PDE4B and PDE4D inhibitor rolipram and PDE inhibitor IBMX were used as a positive control.
Sahin Z., Biltekin S.N., Yurttas L., Berk B., Özhan Y., Sipahi H., Gao Z.G., Jacobson K.A, & Demirayak Ş. (2020). Novel cyanothiouracil and cyanothiocytosine derivatives as concentration-dependent selective inhibitors of U87MG glioblastomas: Adenosine receptor binding and potent PDE4 inhibition. European journal of medicinal chemistry, 212, 113125.
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3

PDE Activity Assay in hiPSC-CM Spheroids

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Pure hiPSC-CM spheroids were pre-treated with COA-Cl or control (PBS) for 30 min. These spheroids were dissociated by incubation with AccuMax for 60 min at 37°C and pipetting every 15 min. After incubation, these cells in 300 μl lysis buffer were briefly centrifuged at 20,000×g for 10 min at 4°C. The lysed cells were then incubated with the substrate cAMP at 37°C for 10 min, and using a PDE activity assay kit (Abcam, Inc., Cambridge, MA), the PDE activity was measured by reading at OD 620 nm on an Envision multi-label plate reader.
Kitsuka T., Itoh M., Amamoto S., Arai K.I., Oyama J., Node K., Toda S., Morita S., Nishida T, & Nakayama K. (2019). 2-Cl-C.OXT-A stimulates contraction through the suppression of phosphodiesterase activity in human induced pluripotent stem cell-derived cardiac organoids. PLoS ONE, 14(7), e0213114.
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4

Quantifying Intracellular cAMP and PDE Activity

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Intracellular cAMP was quantitated using a direct cAMP ELISA kit (Enzo Life Sciences) according to the manufacturer’s instructions. Briefly, cells seeded into plates were scraped and lysed with 0.1 M HCl. Intracellular cAMP levels in the cell lysates were colorimetrically detected at 405 nm using the cAMP ELISA Kit. The final cAMP concentrations were calculated after being normalized by total protein content. For PDE activity assay, cell lysates were centrifuged and the supernatants were collected for further analysis. After purification by gel filtration, the supernatants were detected at 620 nm using a PDE Activity Assay Kit (Abcam) according to the manufacturer’s instructions. IBMX (a potent PDE inhibitor, 10 μM) was applied as the negative control. Quantification of the PDE activity was conducted with drawing a standard curve.
Yu W., Chen F.C., Xu W.N., Ding S.L., Chen P.B., Yang L., Jiang S.D, & Pan X.Y. (2020). Inhibition of Y1 Receptor Promotes Osteogenesis in Bone Marrow Stromal Cells via cAMP/PKA/CREB Pathway. Frontiers in Endocrinology, 11, 583105.
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5

Nicotine Modulates Insulin-Stimulated PDE Activity

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Isolated adipocytes were pre-treated with nicotine (Nic, 300nM) or vehicles (Veh) for 4h. After being incubated with insulin for 10 min, the cells were assayed for PDE activity (PDE Activity Assay Kit, Abcam, ab139460), according to the instruction from the supplier.
Wu Y., Song P., Zhang W., Liu J., Dai X., Liu Z., Lu Q., Ouyang C., Xie Z., Zhao Z., Zhuo X., Viollet B., Foretz M., Wu J., Yuan Z, & Zou M.H. (2015). Activation of AMPKα2 in adipocytes is essential for nicotine-induced insulin resistance in vivo. Nature medicine, 21(4), 373-382.
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6

Uterine Artery Contractility Assay

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Uterine arteries were freshly obtained from nonpregnant mice as described above. Three treatment groups were generated; specifically, untreated control, DA (10 μm, 10 min)-treated, and PTX (750 ng mL−1, 2 h)-treated groups. All incubations were performed in Ca2+-free HEPES buffer at 37°C. After various treatments, the arterial vessel samples were flash-frozen in liquid nitrogen and stored at −80°C for further processing and analysis later. Sample preparation and analysis were conducted in accordance with the protocol provided with the PDE Activity Assay Kit (No. ab241034, Abcam, MA).
Sun B., Smith N., Dixon A.J, & Osei-Owusu P. (2024). Phosphodiesterases Mediate the Augmentation of Myogenic Constriction by Inhibitory G Protein Signaling and is Negatively Modulated by the Dual Action of RGS2 and 5. Function, 5(2), zqae003.
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7

Nicotine Modulates Insulin-Stimulated PDE Activity

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Isolated adipocytes were pre-treated with nicotine (Nic, 300nM) or vehicles (Veh) for 4h. After being incubated with insulin for 10 min, the cells were assayed for PDE activity (PDE Activity Assay Kit, Abcam, ab139460), according to the instruction from the supplier.
Wu Y., Song P., Zhang W., Liu J., Dai X., Liu Z., Lu Q., Ouyang C., Xie Z., Zhao Z., Zhuo X., Viollet B., Foretz M., Wu J., Yuan Z, & Zou M.H. (2015). Activation of AMPKα2 in adipocytes is essential for nicotine-induced insulin resistance in vivo. Nature medicine, 21(4), 373-382.
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8

Uterine Artery Contractility Assay

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Uterine arteries were freshly obtained from nonpregnant mice as described above. Three treatment groups were generated; specifically, untreated control, DA (10 μm, 10 min)-treated, and PTX (750 ng mL−1, 2 h)-treated groups. All incubations were performed in Ca2+-free HEPES buffer at 37°C. After various treatments, the arterial vessel samples were flash-frozen in liquid nitrogen and stored at −80°C for further processing and analysis later. Sample preparation and analysis were conducted in accordance with the protocol provided with the PDE Activity Assay Kit (No. ab241034, Abcam, MA).
Sun B., Smith N., Dixon A.J, & Osei-Owusu P. (2024). Phosphodiesterases Mediate the Augmentation of Myogenic Constriction by Inhibitory G Protein Signaling and is Negatively Modulated by the Dual Action of RGS2 and 5. Function, 5(2), zqae003.
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9

Propofol's Effect on Hippocampal PDE4

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Twenty-four rats, aged 10–12 weeks, were randomly divided into four groups: control group at 30 min (intralipod, 5 ml/kg, i.p.), propofol group at 30 min (25 mg/kg, i.p.), control group at 120 min and propofol group at 120 min. Rats were decapitated 30 min and 120 min after the propofol injection respectively. The CA1 region of hippocampal tissues was harvested in cold ACSF and homogenated in a protein extraction reagent. Protein concentrations were determined by the BCA method according to BCA Protein Assay protocol. PDE4 activity assay was according to the instructions of PDE Activity Assay Kit (Abcam, Shanghai, China).
Li W., Yu L., Yan X., Cai L., Wan L., Teng Q., Li Y., Wang Y, & Xu H. (2018). Reduced Cyclic Adenosine Monophosphate Level in Hippocampal CA1 Participates in Propofol Induced Amnesia in Rats. Frontiers in Neuroscience, 12, 337.
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10

PDE Activity Assay in Rat Brain

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The animal experiments were approved by the Institutional Review Board for animal research and performed according to the guidelines for animal use in laboratories established by Fudan University and Second Military Medical University. All rats, purchased from SLAC laboratory animal company (Shanghai, China), were maintained on a 12 h light/dark cycle with food and water provided ad libitum.
PDE Activity Assay Kit (Colorimetric) was purchased from Abcam Company (Shanghai, China). Forskolin(FSK), 3-Isobutyl-1-methylxanthine(IBMX), Rolipram, Dimethyl sulfoxide (DMSO), N-(2,6 dimethylphenylcarbamoylmethy l), propofol, intralipid and cAMP Enzyme Immunoassay Kit were purchased from Sigma-Aldrich Co. LLC (Shanghai, China). propofol was dissolved in DMSO, and the final concentration was 0.1% in the superfusing artificial cerebrospinal fluid (ACSF). RAMH and the other drugs were administered by addition to ACSF in the in vitro experiment. For the in vivo experiment, propofol (10 mg/ml) was purchased from B. Braun (Shanghai, China).
Li W., Yu L., Yan X., Cai L., Wan L., Teng Q., Li Y., Wang Y, & Xu H. (2018). Reduced Cyclic Adenosine Monophosphate Level in Hippocampal CA1 Participates in Propofol Induced Amnesia in Rats. Frontiers in Neuroscience, 12, 337.
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