The intracellular cAMP level of BFTC905 cells was monitored via the cAMP-GloTM assay by Promega (Madison, WI, United States). BFTC905 were seeded on a 96 well plate (Thermo Fisher Scientific, Waltham, MA, United States) at a density of 20,000 cells per well 1 day prior to the experiment. Cells were washed in induction buffer (Ringer’s solution containing 500 μM IBMX and 100 μM Ro 20-1724) and incubated with Sandranol alone or Sandranol with the specific adenylyl cyclase inhibitor SQ22536 (100 μM, Abcam, Cambridge, United Kingdom) diluted in induction buffer for 20 min. As controls, 0.1% DMSO or 0.1% DMSO with 100 μM SQ22536 were used. The assay was conducted according to the manufacturer’s protocol. Luminescence was monitored via a plate reader (Packard, PerkinElmer, Waltham, MA, United States) and relative light units were normalized to the DMSO (0.1%) control.
Sq22536
Manufactured by Abcam
Sourced in United Kingdom
SQ22536 is a selective inhibitor of adenylate cyclase, an enzyme involved in the production of cyclic AMP (cAMP). It acts by directly inhibiting the catalytic activity of adenylate cyclase, thereby reducing cAMP levels within cells.
Automatically generated - may contain errors
Lab products found in correlation
Camp glotm assay, by Promega (1 mentions)
96 well plate, by Thermo Fisher Scientific (1 mentions)
Chenodeoxycholic acid, by Merck Group (1 mentions)
Ca 074 me, by Merck Group (1 mentions)
2 7 dichlorofluorescein diacetate dcf da, by Thermo Fisher Scientific (1 mentions)
Tyrphostin ag 1478, by Merck Group (1 mentions)
Sp600125, by Cell Signaling Technology (1 mentions)
Z guggulsterone, by Santa Cruz Biotechnology (1 mentions)
Cytochalasin d, by Santa Cruz Biotechnology (1 mentions)
A438079, by Merck Group (1 mentions)
Belnacasan vx 765, by Selleck Chemicals (1 mentions)
U0126, by Cell Signaling Technology (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Mk 2206, by Selleck Chemicals (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharide, by Merck Group (1 mentions)
N acetyl l cysteine, by Merck Group (1 mentions)
Iberiotoxin, by Bio-Techne (1 mentions)
Gallein, by Bio-Techne (1 mentions)
Charybdotoxin, by Bio-Techne (1 mentions)
6 protocols using sq22536
1
Monitoring BFTC905 cAMP Levels
2
Inflammatory Pathways Modulation Protocol
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Lipopolysaccharide (LPS), Chenodeoxycholic acid (CDCA), CA-074 Me, Tyrphostin AG 1478, A438079 and N-acetyl-L-cysteine (NAC) were purchased from Sigma-Aldrich (St. Louis, MO). SQ22536 was obtained from Abcam (Cambridge, MA). Cytochalasin D and Z-Guggulsterone were purchased from Santa cruz Biotechnology (Santa cruz, CA). VX-765 (belnacasan) and MK-2206 were purchased from Selleck ( Houston, TX). SP600125 and U0126 were obtained from Cell Signaling Technologies (Beverly, MA). RPMI 1640, DMEM and antibiotics were obtained from Invitrogen (Carlsbad, CA). 2’, 7’-dichlorofluorescein diacetate (DCF-DA) were from Invitrogen/Molecular Probes. ELISA Kits were purchased from eBioscience (San Diego, CA).
3
Inhibition of Adenylyl Cyclase Regulates cAMP
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Differentiated 3T3-L1 cells were pretreated for 1 h with 0.5% (v/v) DMSO alone (vehicle) or 10 μmol/L of the adenylyl cyclase selective inhibitor SQ22536 (Abcam, Cambridge, UK) dissolved in DMSO and diluted in serum-free DMEM. Differentiated 3T3-L1 cells cultured using the transwell method were treated with butyrate, PGE2, PTX, or SQ22536 for 24 h. Protein was extracted from 3T3-L1 cells cultured in the lower chamber, and cAMP levels were determined using a cAMP Total EIA kit in accordance with the manufacturer’s instructions. The results were standardized by dividing the cAMP concentration by the intracellular total protein concentration (1 mg/mL).
4
Comprehensive Odorant Preparation and Pharmacological Inhibition
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Unless otherwise stated, all odorants of the highest purity available were purchased from Sigma-Aldrich (SI Appendix, Table S1 ). Odorants were freshly prepared on each day of the experiments; they were first reconstituted in DMSO and serially diluted in the appropriate buffer or media. The following pharmacological inhibitors were reconstituted in DMSO, kept at −20 °C, and diluted in the appropriate buffer or media prior to experiments: gallein, U73122, iberiotoxin, charybdotoxin, MRS1845, CaCCinh-A01, H89, CFTRinh-172 (all from Tocris), SQ22536 (Abcam), l -cis diltiazem (Enzo Life Sciences), verapamil (Sigma), and ivacaftor (VX-770, Selleck).
5
Fura-2-AM Intracellular Ca2+ Imaging
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Thyroid cells were incubated for 30 min in loading buffer containing Ringer’s solution (140 mM NaCl, 5 mM KCl, 5 mM CaCl2, 5 mM MgCl2, and 1 mM HEPES) and Fura-2-AM (Invitrogen, Carlsbad, United States). Subsequently, the loading buffer got removed and the cells were rinsed with Ringer solution. The analysis was performed using an Olympus fluorescence microscope (IX71, Olympus, Shinjuku, Japan), a polychromator (MT 20, Olympus), and 10x objective (Olympus). The images were recorded at integrated fluorescence ratios (f340/f380) via the Cell-R Software (Olympus). The analyzed area was randomly selected. The inhibitors: SQ22536 (10 μM, Abcam, Cambridge, United Kingdom), U-73122 hydrate (2 μM, Sigma-Aldrich; St. Louis, United States), and L-cis Diltiazem (100 μM, Abcam) were dissolved in DMSO or aqua dest according to the manufacturer’s instructions.
6
Adipose-Derived Mesenchymal Stem Cell Senescence
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MSCs were isolated from discarded adipose tissue removed from healthy patients undergoing liposuction surgery, as described [22] . Informed consent was obtained from all patients. The procedure was approved by the Ethics Committee of the Chinese Academy of Medical Sciences and Peking Union Medical College. The isolated cells were seeded in T75 asks containing 12 mL culture medium and incubated under humidi ed conditions at 37°C and 5% CO 2 . MSCs were continuously cultured to the third passage and then used for experiments. Cells were either left untreated or treated for 24 h with forskolin (60 µM, Target Mol, Boston, MA, USA). Pyrrolidine dithiocarbamic acid (PDTC, Beyotime, Shanghai, China) 100µM or adenylate cyclase inhibitor SQ22536 (Abcam, Cambridge, UK) 30µM were added to study their effects on MSCs senescence.
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