Glial and Bb cultures were washed in their respective media, devoid of antibiotics. Controls with no spirochetes were also included. Prior to stimulation with live Bb, glial cell cultures were incubated with various concentrations of dexamethasone 5 μM, 15 μM and 150 μM (Sigma) or meloxicam 1 μM, 10 μM, 50 μM and 100 μM (Sigma) for 2 h at 37 °C, after which they were washed and then incubated in fresh growth medium containing the respective concentrations of dexamethasone or meloxicam and live Bb at a multiplicity of infection (MOI) of 10:1 at 37 °C. The effect of the carrier substance of dexamethasone (2-hydroxypropyl)-β-cyclodextrin), at the respective molar concentrations accompanying dexamethasone, was assessed by incubating rhesus astrocytes and microglial cultures in the presence and absence of Bb and carrier alone at 15, 45, and 450 μM, respectively, as previously described for oligodendrocyte cultures [16 (link)]. After 48 h, culture supernatants were collected and processed for evaluation of inflammatory mediators. The drug concentrations were chosen based on previous reports where dexamethasone (5–150 μM) had been shown to inhibit the production of CCL2 in mice microglia [18 (link)], and meloxicam (1–100 μM) had been shown to be effective in inhibiting COX-2 in vitro [19 (link)].
Meloxicam
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom
Meloxicam is a non-steroidal anti-inflammatory drug (NSAID) used as a veterinary medication. It is typically administered orally to reduce inflammation and pain in animals.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (4 mentions)
Esculetin, by Merck Group (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Tenoxicam, by Merck Group (2 mentions)
Piroxicam, by Merck Group (2 mentions)
Probenecid, by Merck Group (2 mentions)
Amphotericin b, by Merck Group (2 mentions)
Ibuprofen, by Merck Group (2 mentions)
Aspirin, by Merck Group (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
Deferoxamine mesylate, by Merck Group (2 mentions)
Zno nanogard, by Thermo Fisher Scientific (2 mentions)
N acetylcysteine, by Merck Group (2 mentions)
Diclofenac sodium salt, by Merck Group (2 mentions)
Ibuprofen, by Promega (2 mentions)
Meloxicam, by Promega (2 mentions)
Ibuprofen i4883, by Merck Group (2 mentions)
Celltiter glo luminescent cell viability assay, by Promega (2 mentions)
Celecoxib, by Merck Group (2 mentions)
43 protocols using meloxicam
1
Dexamethasone and Meloxicam Modulate Glial Inflammatory Response to Borrelia burgdorferi
2
Nociceptive scrunching behavior in planarians
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To induce nociceptive scrunching behavior, we used various concentrations of Allyl isothiocyanate (AITC, CAS:57–06-7, Sigma-Aldrich), ranging from 5 to 200 μM. AITC is an irritant, responsible for the pungency of mustard, horseradish and wasabi, and a potent TRPA1 receptor agonist (Bandell et al., 2004 (link)). It was already used in former studies for its high efficiency to induce scrunching in planarians (Arenas et al., 2017 (link); Sabry et al., 2019 (link)). AITC oil was mixed in DMSO (CAS:37–38-5, Sigma-Aldrich) to allow final solubilization in freshwater. In final concentrations, DMSO never reached more than 0.1%. At these concentrations, DMSO does not induce any toxicity or scrunching on its own (Pagán et al., 2006 (link); Sabry et al., 2019 (link)). The first analgesic we used was morphine HCl (CAS:57–27-2, Francopia) at either 1, 10 or 20 μM, diluted in Volvic mineral water (Volvic, France). The second analgesic we used was meloxicam (sodium salt hydrate form, CAS:71125–39-8, Sigma-Aldrich), a common non-steroidal anti-inflammatory drug (NSAID) at either 1, 10 or 100 μM, also diluted in Volvic water.
3
Amyloid-beta Peptide Aggregation Protocol
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Amyloid-beta peptides (fragment 25–35) were obtained from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in sterile filtered water and aggregated by incubation at 37°C for 4 days before use. Poly(ε-caprolactone) (molecular weight 80.000 g/mol), curcumin, meloxicam and sorbitan monostearate (Span 60®) were obtained from Sigma-Aldrich. High performance liquid chromatography (HPLC) solvents were obtained from JT-Baker (Aparecida de Goiânia, GO, Brazil). All other chemicals were of analytical grade and obtained from standard commercial suppliers (Porto Alegre, RS, Brazil).
4
Synthesis and Characterization of 4-PSCO
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Ethylcellulose polymer (Ethocel®TM Standard 10 Premium) was generously provided by Colorcon (São Paulo, Brazil). Polysorbate 80 (Tween® 80) and medium-chain triglycerides (MCT) were supplied by Delaware (Porto Alegre, Brazil), while sorbitan monooleate (Span® 80) was sourced from Sigma Aldrich (São Paulo, Brazil). Acetone was obtained from CRQ Quimica (São Paulo, Brazil), and high-performance-liquid-chromatography-grade acetonitrile was acquired from Li-Chrosolv (São Paulo, Brazil). All remaining reagents and solvents were of analytical grade and were utilized without further modification. The synthesis and characterization of 4-PSCO (Figure 1 ) were conducted at the Department of Chemistry at the Federal University of Santa Maria [14 (link)]. Analysis of GC/MS determined the chemical purity of this compound (99.9%). The 4-PSCO was dissolved in canola oil. Morphine and Meloxicam (used as reference drugs) were dissolved in an isotonic saline solution. Meloxicam and morphine (Sigma Aldrich, São Paulo, Brazil) were administered intragastrically (i.g.) and intraperitoneally (i.p.), respectively, at a constant volume of 10 mL/kg of body weight.
5
In vivo Stereotaxic Injection of Lentiviral Vectors
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Viral titers of LacZ: 1.7 × 107 RIU/ml, GSK3α: 3 × 107 RIU/ml and GSK3β: 2.5 × 107 RIU/ml (RNAi core, Academia Sinica, Taiwan), were used for in vivo stereotaxic injections. Lentivirus was thawed on ice and resuspended by pipetting before use. A mixture of 80 mg/kg ketamine and 40 mg/kg xylazine solution was intraperitoneally injected to anesthetize the animals. Lentivirus was stereotaxically injected into the mPFC using a 30-gauge blunt-tip needle connected to a syringe (Hamilton Robotics, Reno, NV, USA) by a poly-ethylene catheter. The stereotaxic coordinate for the mPFC was AP: + 1.9 mm, ML: 0.5 mm, DV: − 0.28 mm relative to the bregma. Each mouse was injected bilaterally (1 μl each side), receiving vectors encoding shRNA directed against GSK3α, GSK3β or LacZ. Meloxicam (Sigma Aldrich, St. Louise, MO, USA) was administered (5 mg/kg, s.c.) for 2 days after surgery to relieve pain from the surgical operation. The NOR task was carried out 7 days after surgery.
6
In vitro blood-brain barrier assay
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Celecoxib, diclofenac, lornoxicam and diazepam were a kind gift of Dr. Maierhofer (AGES, PharmMed, Austria), whereas ibuprofen (I1892, SigmaAldrich, Austria), meloxicam (M3935, SigmaAldrich, Austria), piroxicam (P5654, SigmaAldrich, Austria), tenoxicam (T0909, SigmaAldrich, Austria), carboxyfluorescein (21877, Fluka, Switzerland), probenecid (P8761, SigmaAldrich, Austria) and verapamil (94837, Fluka, Switzerland) were purchased from commercial sources. Iscove’s modified Dulbecco’s medium (IMDM), Ham reg. nutrient mixture F12 (Ham’sF-12), newborn calf serum (NCS), l-glutamine and penicillin/streptomycin were obtained from Invitrogen Life technologies (GibcoTM, Carlsbad, CA). Heparin and collagen solution (predominantly collagen I, 150703) were purchased from MP Biomedicals (Irvine, CA). Amphotericin B, transferrin, 8-aminopyrene-1,3,6-trisulfonate (APTS) and gelatine were from SigmaAldrich (Austria), whereas dextran (av. MW 6000) was from Fluka (Switzerland). Fibronectin was obtained from BD Biosciences (Bedford, MA) as well as Transwell inserts (FalconTM) and six-well plates (FalconTM). Basal endothelial and astrocyte media and components for primary rat endothelial cells and astrocytes were provided from Biopredic Int. (France). Inorganic salts and all other reagents were of analytical grade.
7
Purification and Analysis of ALDH2
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Tunicamycin, 4-phenylbutylate, cycloheximide, ibuprofen, and lysozyme were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). The ALDH2 protein (18–517 aa) was obtained from ATGen (Seoul, Korea). (±)-Flurbiprofen were from Sigma (St Louis, MO, USA) or Cayman Chemical (Ann Arbor, MI, USA). α-Lactalbumin, aspirin, and meloxicam were from Sigma. Human recombinant leptin for use in vitro was from Sigma and mouse recombinant leptin for use in vivo was from R'D Systems (Minneapolis, MN, USA). Ferriteglycidyl methacrylate (FG) beads (epoxy beads: TAS8848N1110) were from Tamagawa Seiki (Tokyo, Japan). 4′-hydroxy flurbiprofen was obtained from Toronto Research Chemicals (Toronto, ON, Canada).
8
Protein-Polyelectrolyte Interaction Assay
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Polyelectrolytes (PAA, 1800 g/mol, ≥99%), HSA (66 kDa)); fluorescence probe (pyrene, ≥99%), and drugs (warfarin (≥98.0%), meloxicam, amphotericin B) were purchased from Sigma–Aldrich (St. Luis, MO, USA) and used as received. The PS-16 and IA-16 were synthesized similarly to the procedure indicated in refs. [48 (link),49 (link)], respectively.
9
Analytical Techniques for Compound Characterization
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Melting points were determined on an Electrothermal apparatus. Optical rotations were measured on a JASCO V-550 UV/Vis spectrometer (Tokyo, Japan). The NMR [1H (500 MHz), 13C (125 MHz)] experiments were performed on a Bruker Advance 500 spectrometer (United State). Chemical shift was reported in ppm downfield from TMS, with J in Hz. Mass spectra were obtained with an AGILENT 1200 series LC-MSD Ion Trap (United State). Analytical TLC was performed on Kieselgel 60 F254 (Merck) plates (silica gel, 0.25 mm layer thickness) and RP-18 F254 (Merck) plates (0.25 mm layer thickness). UV spots were visualized using ultraviolet irradiation (at 254–365 nm) and by spraying with 10% H2SO4, followed by heating with a heat gun. Column chromatography was performed on silica gel (70–230 and 230–400 mesh, Merck), YMC RP-18 resin (30–50 μm, Fuji Silysia Chemical Ltd.), and Sephadex™ LH-20 columns (Amersham Biosciences, Uppsala, Sweden). Dulbecco’s modified Eagle’s medium (DMEM), trypsin and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY). COX-2 (1:1000, Cat: 610204) was obtained from BD Biosciences. Secondary mouse or rabbit HRP-conjugated antibodies (1:5000 or 1:10,000, Cell Signaling, #7074, #7076). β-actin (Cat: A5316), LPS (Cat: L4391), meloxicam (cat: M3935) and MTT (cat: M2128) were purchased from Sigma-Aldrich.
10
Canine Cell Line Culturing and Immune Modulation
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The canine melanoma cell lines CMeC, LMeC, CMM-1, and CMM-232 (link),33 (link), were cultured as described previously6 (link). The canine osteosarcoma cell lines POS34 (link) and HMPOS35 (link) were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 200 μg/mL streptomycin, and 200 U/mL penicillin (Thermo Fisher Scientific) at 37 °C under a 5% CO2 atmosphere. Canine PBMCs were purified from heparinized blood obtained from healthy beagles by density gradient centrifugation on Percoll (GE Healthcare UK, Buckinghamshire, UK) and cultured as described previously6 (link). PBMCs were stimulated with 5 μg/mL Staphylococcal Enterotoxin B (SEB) (Sigma-Aldrich) and 1 μg/mL anti-canine CD28 antibody (eBioscience, San Diego, CA). In some experiments, cells were co-treated with 5 μM meloxicam (Sigma-Aldrich), 2.5 μM Prostaglandin E2 (Cayman Chemical), and/or 20 μg/mL canine chimeric anti-PD-L1 antibody c4G1211 (link) as indicated. The same concentrations of DMSO (Nacalai Tesque, Kyoto, Japan) and dog IgG (Jackson ImmunoResearch, West Grove, PA) were used as negative controls.
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