Capsaicin was dissolved in ethanol (Sigma) and prepared in serum-free medium containing medium-199 and antibiotics/antimycotic (SF) at concentrations of 50, 125, 250 and 500 µM for experiments Formoterol and roflumilast (Tocris, Medsantek, Türkiye) were dissolved in dimethyl sulfoxide (DMSO), and concentrations of 1 μM formoterol and 0.1 µM roflumilast were added to solutions containing 250 μM and 500 µM capsaicin for CBF and TEER experiments, respectively. These specific concentrations of capsaicin (Tsukura et al., 2007 (link)), formoterol (Erjefält and Persson, 1991 (link); Anderson, 1993 (link)), and roflumilast (Milara et al., 2012 (link); Schmid et al., 2015 (link)) were chosen based on the existing literature, and our ongoing studies investigating effects of formoterol and roflumilast on CBF in primary BECs.
Roflumilast
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, India
Roflumilast is a selective and competitive phosphodiesterase-4 (PDE4) inhibitor. It is used in the development and manufacturing of various laboratory and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Rolipram, by Merck Group (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Tadalafil, by Merck Group (2 mentions)
Wortmannin, by Merck Group (2 mentions)
Caffeine, by Merck Group (2 mentions)
Forskolin, by Merck Group (2 mentions)
Cresyl violet, by Merck Group (2 mentions)
Anti pde4b nb100 2562, by Novus Biologicals (2 mentions)
Camp elisa kit, by Cayman Chemical (2 mentions)
Congo red, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Cigarette smoke generator, by Daehan Biolink (1 mentions)
Ethanol, by Merck Group (1 mentions)
Carboxymethylcellulose, by Merck Group (1 mentions)
Rotenone, by Merck Group (1 mentions)
Kt5720, by Merck Group (1 mentions)
Rp camps, by Biolog (1 mentions)
R rolipram, by Bio-Techne (1 mentions)
Isoproterenol, by Merck Group (1 mentions)
Dl propargylglycine, by Merck Group (1 mentions)
19 protocols using roflumilast
1
Capsaicin, Formoterol, and Roflumilast Interactions
2
Cigarette Smoke-Induced COPD Alleviation
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The CS was generated from 3R4F research cigarette (Kentuchy reference cigarette, University of Kentuchy, USA), containing 11.0 mg of total particulate matter, 9.4 mg of tar, and 0.76 mg of nicotine per cigarette. Exposure to CS (one puff/min, 35 mL puff volum over 2 seconds, every 60 seconds, 8 cigarettes per day) was conducted using cigarette smoke generator (Daehan Biolink, Republic of Korea). The mice were exposed to CS for 1 h in a chamber (50 cm×30 cm×30 cm) for 28 days. LPS were intranasally instilled (10 µg dissolved in 50 µL distilled water) under anesthesia on day 26. The HemoHIM was obtained from Korea Institute of Oriental Medicine (Daejeon, Republic of Korea). HemoHIM was administered to mice at doses of 50 or 100 mg/kg by oral gavage 1 h before CS exposure for 28 days. A positive control group was administered roflumilast (Sigma-Aldrich, St, Louis, MO, USA, 10 mg/kg) which is a PDE-4 inhibitor and manufactured for treatment COPD.
3
Rotenone and Roflumilast Preparation Protocol
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Rotenone and roflumilast were purchased from Sigma-Aldrich (St. Louis, MO, USA), while dimethyl sulfoxide (DMSO) and carboxymethylcellulose (CMC) were obtained from Merck (Darmstadt, Germany) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. Levodopa was acquired from Merck and Co. Inc. (New Jersey, USA). All used chemicals were of the highest purity and analytical grade. Rotenone was prepared in 1% DMSO, while roflumilast was suspended in 1% CMC.
4
Pharmacological Inhibition of Rho Signaling
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The compounds used and their suppliers are listed below (Table 1 ): a cell-permeable version of Clostridium botulinum-derived Rho inhibitor, C3 ADP-ribosyltransferase (C3, Cytoskeleton); the ROCK inhibitor, Y27632 (Calbiochem, Merck Millipore), the PDE4 inhibitors, rolipram (Sigma), R-rolipram and S-rolipram (Tocris Bioscience, R & D Systems), which specifically inhibit the HARBS and LARBS respectively. RS25344 hydrochloride (Tocris) and roflumilast (Sigma), which like rolipram inhibit both HARBS and LARBS; the PKA inhibitors KT-5720 (Sigma) and Rp-cAMPS (Biolog), and the Epac agonist 8-pMeOPT-2′-O-Me-cAMP (Me-cAMP; Biolog, Bremen, Germany).
5
Bladder Neck Contractility Assay
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The drugs used were: Isoproterenol, NG-nitro-L-arginine (L-NOARG), phenylephrine (PhE), DL-propargylglycine (PPG), roflumilast and tadalafil, all from Sigma (USA). PPG and roflumilast were dissolved in dimethylsulphoxide. tadalafil was dissolved in acetonitrile. The other compounds were dissolved in double-distilled water. Preliminary experiments revealed no effects of the solvents used on bladder neck samples contractility.
PSS composition (in mM)was: NaCl 119, KCl 4.6, MgCl2 1.2, NaHCO3 24.9, glucose 11, CaCl2 1.5, KH2PO4 1.2, ethylenediamine tetraacetic acid (EDTA) 0.027. PSS was maintained at 37 °C and gassed with 95% O2 and 5% CO2, thus obtaining a final pH of 7.4 in the organ bath. To prepare K+-enriched PSS (KPSS), the NaCl in PSS was replaced with KCl on an equimolar basis.
PSS composition (in mM)was: NaCl 119, KCl 4.6, MgCl2 1.2, NaHCO3 24.9, glucose 11, CaCl2 1.5, KH2PO4 1.2, ethylenediamine tetraacetic acid (EDTA) 0.027. PSS was maintained at 37 °C and gassed with 95% O2 and 5% CO2, thus obtaining a final pH of 7.4 in the organ bath. To prepare K+-enriched PSS (KPSS), the NaCl in PSS was replaced with KCl on an equimolar basis.
6
Modulating cAMP Signaling in Immune Cells
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Dibutyryl-cAMP, rolipram, roflumilast, Escherichia coli LPS (055:B5), goat γ-globulin, 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and actinomycin D were obtained from Sigma-Aldrich (MO, USA), and the CREB inhibitor C217505 was from Merck Millipore (MA, USA). The PKA activator N6-benzoyladenosine-3′, 5′-cyclic monophosphate (6-Bnz-cAMP), Epac activator 2′-O-methyladenosine-3′, 5′-cyclic monophosphate (8-pCPT-2′-O-Me-cAMP), and PKA inhibitor 8-(4- Chlorophenylthio)-8-(4-Chlorophenylthio) adenosine-3′, 5′-cyclic monophosphorothioate, Rp-isomer (Rp-8-CPT-cAMPS) were purchased from BioLog Life Science Institute (Bremen, Germany). The RPMI 1640 and DMEM medium base and fetal bovine serum (FBS) were obtained from Thermo Fisher Scientific (MA, USA).
7
HBV Entry Inhibitors and Molecular Probes
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Paraformaldehyde (PFA), Triton™ X-100, Tunicamycin, Cytochalasin D, Latrunculin A, Colchicine, Demecolchine, Taxol, cAMP, Wortmannin, MDL12, Caffeine and Roflumilast were obtained from Sigma-Aldrich. DMSO was from VWR International. For detection of the NTCP protein, an anti-FLAG antibody (DYKDDDDK Epitope Tag antibody; ThermoFisher Scientific) was used, while for the detection of HBV core particles, an anti-HBcAg was ordered from Dako, Agilent Technologies Antibodies against β-actin and γ-Tubulin were from Sigma-Aldrich, while PCNA was obtained from Abcam. Small interference RNAs (siRNAs) were ordered from ThermoFisher Scientific. cAMP direct immunoassay kit was purchased from Abcam. Human phospho-kinase arrays were performed according to the manufacturer's instructions (R&D Systems).
8
CD8 cell stimulation with dexamethasone
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PBMCs and peripheral blood CD8 cells were seeded in triplicate in 96 well plates at 100,000 cells per well. Pulmonary CD8 cells were seeded at 50,000 cells per well. Cells were pre-treated for 1 h with dexamethasone (10−6 M – 10−10 M) alone or in combination with GSK256066 (Stratech, Newmarket, UK, Spain), roflumilast (Sigma-Aldrich, Poole, UK) or forskolin (Sigma-Aldrich). Cells were then stimulated for 24 h with anti-CD2/3/28 beads (Miltenyi Biotec). Supernatants were harvested and stored at −20 °C until ELISA was performed.
9
Pharmacological Screening Compound Acquisition
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Leflunomide, teriflunomide, tolvaptan and BTP2 were purchased from Tocris Bioscience (Bristol, UK). Omeprazole, lansoprazole, rufinamide, prazosin hydrochloride, terazosin hydrochloride, flutamide, roflumilast and propidium iodide were purchased from Sigma-Aldrich (Dorset, UK). Conivaptan hydrochloride was purchased from Selleckchem (distributed by Stratech, Suffolk, UK) and OAG was purchased from Cayman Chemicals (distributed by Cambridge Bioscience, Cambridge, UK). All molecules were of ≥98% purity certified by the vendors.
10
Phytochemicals Modulate COPD in Mice
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C57BL/6N male mice (6-week-old, 20-25 g) were obtained from Koatech Co., (Pyeongtaek, Korea) and were provided sterilized tap water and commercial rodent chow (Samyang Feed Co., Wonju, Korea). All procedures were granted by the Institutional Animal Care and Use Committee of Chonnam National University (Gwangju, Korea).
CS exposure was performed as previously described [12 (link)]. LPS (10 µg/mouse, Sigma-Aldrich, MO, USA) was treated by intranasal instillation on day 12 and 26 under anesthesia. Roflumilast (Sigma-Aldrich) was a phosphodiesterase-4 inhibitor recommended for treating COPD and used as a positive control drug [13 (link)]. Roflumilast (10 mg/kg) and PYC (10 and 20 mg/kg, Horphag Research, Le Sen, France) were administered to animals for 4 weeks by oral gavage 1 h before the CS exposure. Tentative identification of phytochemicals in PYC was described in our previous study [12 (link)]. Briefly, five phytochemicals (procyanidin B-type dimer, procyanidin dimer gallate ester, procyanidin B-type trimer, taxifolin-3-O-β-glucoside, and taxifolin) were identified in PYC.
CS exposure was performed as previously described [12 (link)]. LPS (10 µg/mouse, Sigma-Aldrich, MO, USA) was treated by intranasal instillation on day 12 and 26 under anesthesia. Roflumilast (Sigma-Aldrich) was a phosphodiesterase-4 inhibitor recommended for treating COPD and used as a positive control drug [13 (link)]. Roflumilast (10 mg/kg) and PYC (10 and 20 mg/kg, Horphag Research, Le Sen, France) were administered to animals for 4 weeks by oral gavage 1 h before the CS exposure. Tentative identification of phytochemicals in PYC was described in our previous study [12 (link)]. Briefly, five phytochemicals (procyanidin B-type dimer, procyanidin dimer gallate ester, procyanidin B-type trimer, taxifolin-3-O-β-glucoside, and taxifolin) were identified in PYC.
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