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7 protocols using β pea

1

Receptor Binding Assay with TAAR1 Antagonist

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β-PEA and tyramine were purchased from Sigma (St Louis, MO, USA). MA and LSD were generously provided by NIDA Drug Supply Program. Polyethylenimine (PEI, MW 40000) was purchased from Polysciences (Warrington, PA, USA). For experiments in vitro, the TAAR1 antagonist, N-(3-Ethoxy-phenyl)-4-pyrrolidin-1-yl-3-trifluoromethyl-benzamide (EPPTB) [26 (link)] was first diluted in DMSO, and subsequently diluted into cAMP assay buffer for a final DMSO concentration of 0.1%.
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2

Pharmacological Modulation of Dopamine

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β-PEA and a DAD1R antagonist, SCH23390, were purchased from Sigma-Aldrich (St. Louis, MO, USA). β-PEA and SCH23390 were dissolved in 0.9% physiological saline. The intraperitoneal (i.p.) injection for SCH23390 was determined based on a previous study [63 (link)]. For self-administration studies, a working solution of saline or β-PEA was filtered through a syringe-mounted 0.22-µm Minisart® Syringe Filter (Sartorius Stedim Biotech, Goettingen, Germany) immediately before use. All drug solutions were prepared immediately prior to the beginning of each experiment.
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3

Synthesis and Characterization of Psychoactive Compounds

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Bupropion hydrochloride and 6-hydroxybupropion were obtained from Toronto Research Chemicals (Toronto, Canada), ibogaine from Ibogaworld, cocaine from Medisca (Montreal, Canada), β-PEA, pinoline, indole, tryptamine, ephedrine, frovatriptan, alprenolol, isoproterenol, evodiamine, and tyramine from Sigma (Oakville, Canada), fluoxetine and amphetamine were from Tocris Bioscience (Bristol, United Kingdom). PAL analogs, RTI analogs, and bicifadine were synthesized at RTI in the Blough laboratory as described previously (Carroll et al., 2009 (link), 2011 (link); Blough et al., 2011 , 2014 (link)). Noribogaine (Batch No. 606950002) was from Deborah Mash, Ph.D. (Obach et al., 1998 ). Ibogamine was obtained from Specs (Zoetermeer, The Netherlands). Sodium phenylbutyrate was purchased from Enzo Life Sciences Inc. (Farmingdale, NY). Ibogaminalog, ibogainealog, noribogainalog, fluorogainalog, and tabernanthalog were synthesized in the lab of David E. Olson (Cameron et al., 2020 (link)).
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4

Pharmacological Modulation of Social Interaction

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The drug MDMA was dissolved in physiological saline (in a volume of 2 ml/kg) and administered at doses of 0.5, 2.5, and 5 mg/kg. Morphine (ICN Hungary Plc.) was also dissolved in physiological saline (in a volume of 2 ml/kg) and administered at a dose of 1 mg/kg 30 min before the social interaction assay. GBR‐12909 (Sigma) was dissolved in physiological saline (in a volume of 5 ml/kg) and administered at doses of 1, 3, and 10 mg/kg. All the drugs were administered by subcutaneous (s.c.) injection in the flank region 30 min prior to the behavioral observations. In the coadministration experiment, MDMA and GBR‐12909 were administered to the rats in the two flank regions within a short period of time 30 min prior to the observations. DA‐3,4‐[7,8‐3H(N)], with a specific activity of 60 Ci/mmol, and choline chloride [methyl‐3H], with a specific activity of 80 Ci/mmol, were purchased from American Radiolabeled Chemicals (ARC). β‐PEA was obtained from Sigma. All the other chemicals were obtained from Sigma.
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5

Measuring Intracellular cAMP in Astrocytes

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Intracellular cAMP levels in astrocytes were measured using a commercially available homogenous, bioluminescent assay, cAMP-Glo Assay, (Promega Corp., Madison, WI). Adherent monolayers of astrocytes cultured in 96-well plates (50,000 cells/well) were exposed to forskolin, METH and β-PEA (all from Sigma-Aldrich). Cells were directly activated and lysed in the tissue culture plate. Lysates were diluted to a final cell concentration of approximately 1,000 cells/μL in lysis buffer [500 μM 3-isobutyl-1-methylxanthine (IBMX) and 100 μM Ro 20-1724, cAMP specific phosphodiesterase inhibitors (both from Sigma-Aldrich)] and transferred to a white opaque flat bottom 96-well assay plates (Corning Inc. Life Sciences, Tewksbury, MA). Intracellular cAMP levels were assayed using GloMax 96 Microplate Luminometer with dual injectors (Promega). Data analysis for the half-maximal effective concentrations (EC50) was performed with Prism V6.0 (GraphPad Software, La Jolla, CA) using sigmoidal dose-response (variable slope) curve.
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6

Quantification of Cannabinoids and Polyamines

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Deuterated water (D2O) 99.97 atom% of deuterium and 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid sodium salt (TSP) were purchased from Euriso-Top (Saclay, France). HPLC-PDA chemical standards, n-hexadecane, HIS, SPM, SPD, PUT, (β-PEA), CAD, TYM and 1,7-diaminoheptane were purchased from Sigma-Aldrich (Milan, Italy). Methanol (HPLC-grade), chloroform (HPLC-grade), ethanol (analytical-grade), perchloric acid (70%), acetone (analytical-grade), acetonitrile (HPLC-grade) were obtained from Carlo Erba Reagenti (Milan, Italy). Double-distilled water was obtained using a Millipore Milli-Q Plus water treatment system (Millipore Bedford Corp., Bedford, MA, USA). Sodium carbonate (Na2CO3; 99.999% purity), Folin-Ciocalteu’s phenol reagent, tannic acid (Ph. Eur. purity) and aluminium chloride hexahydrate (AlCl3 × 6 H2O; Ph. Eur. purity) were purchased from Merck (Darmstadt, Germany).
Cannabinoids reference standards in methanol CBDV (1 mg/mL), CBG (1 mg/mL), CBD (1 mg/mL), CBN (1 mg/mL), (–)-Δ9-THC (0.1 mg/mL) and CBC (1 mg/mL) with purity ≥99%, were purchased from Cerilliant Corporation (Round Rock, TX, USA). For mobile phase, gradient grade water (H2O) and acetonitrile (ACN) were purchased from Sigma Aldrich (St. Louis, MO, USA) as well as trifluoroacetic acid (TFA) and analytical grade ethanol used for the extraction procedure. All solvents were further filtered on a 0.2 μm filter.
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7

Pharmacological Modulation of Neurotransmitter Signaling

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The drugs used were the following: Klamin® extract (kindly supplied by Nutrigea Research s.r.l., Borgo Maggiore, Republic of San Marino), TTX (Alomone Labs, Jerusalem, Israel), EPPTB (N-(3-Ethoxy-phenyl)-4-pyrrolidin-1-yl-3-trifluoromethyl-benzamide) (Hoffmann-La Roche, Monza, IT, Italy), β-PEA (β-phenylethylamine), L-NAME, 5-HT (Serotonin), methysergide (Sigma-Aldrich, Milano, IT, Italy). All the drugs were dissolved in distilled water. Klamin® extract was dissolved in 0.5 mL water, and then sonicated (twice for 60 s) immediately prior to add to the bath. Chemicals were prepared as stock solution, which were diluted with Krebs solution on the experiment day.
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