Drugs were prepared as 1000× stocks and stored at −20°C. To induce homeostatic scaling, neurons were treated with 20 μm bicuculline methobromide (BCC; Tocris, 20 mm stock in water) or 1 μm tetrodotoxin citrate (TTX; Abcam, 1 mm stock in water) for 48 h (Fig. 1 ). In some live-cell imaging experiments, 20 μm BCC or 1 μm TTX was applied acutely for 10–60 min. For Figure 2 , neurons were treated with 20 μm BCC for 4, 12, 24, or 48 h to monitor the process of homeostatic down-scaling. For Figure 3 , neurons were treated for 48 h with a combination of 1 μm MTEP hydrochloride (Tocris, 1 mm stock in DMSO) and 100 nm JNJ 16259685 (Tocris, 100 μm stock in DMSO) to block mGluR1/5 signaling. For Figure 4 , neurons were treated for 48 h with 100 nm PF3845 (Cayman, 100 μm stock in DMSO) or 2.5 μm PF3845 (Cayman, 2.5 mm stock in DMSO) to inhibit FAAH and to elevate AEA and related NAEs. For Figure 5 , neurons were treated for 6 h with 50 nm AM251 (Cayman, 50 μm stock in DMSO), or for 10–60 min with 500 nm AM251 (Cayman, 500 μm stock in DMSO) to block CB1 signaling. For Extended Data Figure 5-1 , neurons were treated for 10–60 min with 50 μm D-AP5 (Cayman, 50 mm stock in water) to block NMDA signaling.
Am251
Manufactured by Cayman Chemical
Sourced in United States
AM251 is a cannabinoid receptor antagonist that selectively binds to the CB1 receptor. It is commonly used in research applications to investigate the endocannabinoid system.
Automatically generated - may contain errors
Lab products found in correlation
Win55 212 2, by Cayman Chemical (8 mentions)
Urb597, by Cayman Chemical (7 mentions)
Am630, by Cayman Chemical (6 mentions)
Jzl184, by Cayman Chemical (5 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Capsaicin, by Merck Group (3 mentions)
Sr141716a, by Cayman Chemical (2 mentions)
Urb597, by Merck Group (2 mentions)
Sr144528, by Cayman Chemical (2 mentions)
Phosphate salts, by Sartorius (2 mentions)
Pf3845, by Cayman Chemical (2 mentions)
Sb203580, by MedChemExpress (2 mentions)
Sp600125, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Fura 2 am, by Thermo Fisher Scientific (2 mentions)
Hbmscs, by Merck Group (2 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions)
Jwh 018, by Cayman Chemical (1 mentions)
Celltiter 96 non radioactive cell proliferation assay kit, by Promega (1 mentions)
Dulbecco modified eagle medium (dmem), by Atlanta Biologicals (1 mentions)
37 protocols using am251
1
Homeostatic Scaling of Neuronal Activity
2
Cannabinoid Dose Conversion for Humans
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Δ9-THC was provided by Dr. S. Morimoto (Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan) and stored in ethanol at −80 °C. JWH-018, metabolites of JWH-018, deuterium-labelled JWH-018 (JWH-018-d9) and AM-251 were purchased from Cayman Chemical (Ann Arbor, MI, USA) and stored at −20 °C. The doses of cannabinoids presently used here were selected based on the CB1R affinity of the ligands and the electrographic representation of mild seizure events. In fact, we intentionally selected relatively high doses that we knew to have an effect on EEG power spectra39 (link). The conversion of an animal to human dose cannot be done directly, as in the few available studies on human volunteers most utilize the inhalation route, while we administered drugs intraperitoneally. However, after applying the human estimation dose conversion40 (link), 2.5 mg/kg of JWH-018 in mouse corresponds to 0.2 mg/kg, or roughly 12 mg per person. Similarly, 10 mg/kg of ∆9-THC in mice converts to 0.8 mg/kg in humans or about 49 mg per individual.
3
Endocannabinoid Receptor Modulation in Cell Proliferation
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The following were purchased from Cayman (Ann Arbor, MI): 2-AG; the endocannabinoid receptor inhibitors, SR141716A (Rimonabant, CB1 inverse agonist), AM251 (CB1 antagonist) and SR144528 (CB2 inverse agonist); the endocannabinoid receptor activators, CP47497 (CB1 agonist), AM1241 (CB2 agonist), Win- 55–212-2 (CB1 and CB2 agonist) and CP55940 (CB1 and CB2 agonist); the selective and nonselective inhibitors for cyclooxygenase-1 and -2 (COX-1, COX-2), SC-560 (selective for COX-1), CAY10404 (selective for COX- 2), Ibuprofen (nonselective for COX-1 and COX-2); and the selective inhibitors for hydrolases of 2-AG, JZL 184 (selective for enzyme monoacylglycerol lipase, MAGL. Anti-bromodeoxyurine (BrdU) antibody was purchased from Roche Applied Science (Indianapolis, IN). The CellTiter 96 Non-Radioactive Cell proliferation Assay Kit™ was purchased from Promega (Madison, WI). Charcoal/dextran-treated fetal bovine serum (CFBS) was purchased from Hyclone (Logan, UT). DMEM, RPMI-1640, antibiotics (penicillin and streptomycin), and fetal bovine serum (FBS) were purchased from Atlanta Biologicals (Lawrenceville, GA).
4
CB1R Agonist and Antagonist Study
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The CB1R antagonist AM-251 (71670, Cayman Chemicals, Michigan, USA) and agonist WIN55,212-2 (10009023, Cayman Chemicals, Michigan, USA) were given intra-peritoneally.
5
Neuroprotective Effects of PEA Formulations
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In both cell types, cells were treated with (0.1 µM) PEA dissolved in FM-LipoMatrix® (a new technology based on solvent patent N°102017000036744 of noiVita srls, produced by Pro-Bio INTEGRA srl, Rovigo, Italy) in the presence or absence with lipoic acid (50 µM) plus vitamin D3 (100 nM) prepared directly into medium of stimulation (named LA + vitD) [39 (link)]. To verify the effectiveness of this new product the effects were compared to the classic commercial product (0.1 µM micronized PEA) and the solvent technology was also tested alone. The concentration of PEA was obtained by literature to maintain the antioxidant effect [53 (link)] and confirmed by dose-response study (data not shown) [54 (link)]. To verify the mechanism involved, some experiments were carried out also in presence of 30 min pre-treatments with both 10 µM AM251 and 10 µM AM630 (Cayman Chemical Company, Ann Arbor, MI, USA) [55 (link)], the specific CB1 and CB2 inhibitors, respectively. Finally, Lipopolysaccharides (LPS, Sigma-Aldrich, Milan, Italy) 500 ng/mL was used to verify the neuroprotective properties, pre-treating the cell for 24 h [56 (link)].
6
Cannabinoid Receptor Agonists and Inhibitors
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CB1 receptor agonist WIN 55212-2 (WIN, Tocris, 0.1 and 0.5 mg/kg body weight for the intraperitoneal injection, 1 and 2 μg/μl for micro-infusion into the vHip) (Martellotta et al., 1998 (link); Shen et al., 2019 (link)) was dissolved in 50% DMSO (Sigma-Aldrich) and 50% normal saline solution. Arachidonylcyclopropylamide (ACPA, Abcam, 5 or 10 mg/kg body weight for the intraperitoneal injection, 4 ng/μl for micro-infusion into the vHip) (Jafari-Sabet and Karimi, 2017 (link); Shafaroodi et al., 2004 (link); Srisai et al., 2017 (link)) was dissolved in 100% Tocrisolve 100 (Tocris). The FAAH inhibitor URB597 (5 ng/μl, Sigma-Aldrich) (Haller et al., 2009 (link)) was dissolved in 5% DMSO and 95% normal saline solution. MAGL inhibitor JZL184 (2 μg/μl, Tocris) (Folkes et al., 2020 (link)) was dissolved in 1:1 DMSO and Tween 80 (Sigma-Aldrich) and diluted by the normal saline solution to 2% DMSO and 2% Tween 80. The CB1 receptor antagonist AM251 (2.5 ng/μl, Cayman) (Shen et al., 2019 (link)) was dissolved in 25% DMSO and 75% normal saline solution. Drug doses were based on the literature and on pilot experiments. Solutions were freshly prepared and were administrated at a volume of 0.1 ml for the intraperitoneal injection and 0.8 μl per side for the infusion into the vHip and dorsal striatum.
7
Endocannabinoid Receptor Antagonists and Antioxidants
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Endocannabinoids - docosahexaenoyl ethanolamide (DHEA), eicosapentaenoyl Ethanolamide (EPEA) and N-arachidonoyl-L-alanine (NALA), antagonists of CB1 and VR1 (AM251, cay10448), antioxidants (NAC and GSH), and inhibitors of 5-LO (AA861, zileuton and ebselen) were obtained from Cayman Chemical (Ann Arbor, MI).
8
Inhibitors and Agonists for Cell Signaling
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PI3K inhibitor LY294002, IKK inhibitor BMS345541, MEK inhibitor U0126, MAPK p38 inhibitor SB203580 and JNK inhibitor SP600125 were purchased from Calbiochem (La Jolla, CA, USA). Human soluble Enzo Killer-TRAIL (recombinant #ALX-201-073-C020), Super-Fas-Ligand (recombinant; #ALX-522-020) and anti-human TRAIL Ab (#ALX-210-732) were purchased from Enzo Life Sciences (San Diego, CA, USA). Recombinant human TNFα (#210-TA-010) and anti-human TNFα mAb (#MAB610-100) were obtained from R&D Systems (Minneapolis, MN). Anti-human IL6 mAb (#MAB2061R) were purchased from R&D Systems (Minneapolis, MN). Cannabidiol (exempt preparation; #90081); AM251 (#71670); SR144528 (#192703-06-3) and NS398 (#70590) were obtained from Cayman Chemical (Ann Arbor, MI).
9
Cold Exposure and Appetite Regulation
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Following a 4-week acclimatization period, the mice were ranked by weight and distributed into four groups (6 mice/group) of equal mean body weight. Two groups were exposed to 4°C for 4 hours three times per week while 2 control groups were treated similarly at 22°C (control: CON). Mice were treated daily with either vehicle or AM251 (3 mg/kg in 5% DMSO/5% Tween 80 in saline; Cayman Chemical, Ann Arbor, MI) by oral gavage. Body weight and food intake were measured daily prior to gavage.
At the end of the studies, mice were administered ketamine (100 mg/kg) and xylazine (10 mg/kg) by ip injection, blood was obtained by retro-orbital bleed, and tissues were removed, weighed, and frozen at −80°C until assayed.
At the end of the studies, mice were administered ketamine (100 mg/kg) and xylazine (10 mg/kg) by ip injection, blood was obtained by retro-orbital bleed, and tissues were removed, weighed, and frozen at −80°C until assayed.
10
Nanoparticle Formulation for Cannabinoid Delivery
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Rimonabant hydrochloride was purchased from Glentham (Cat #GP8358). PLGA, which has a lactide-glycolide ratio of 50:50 and a MW of 30,000–60,000 (acid-terminated), was purchased from Lactel Absorbable Polymers (Cat #B6013–2). PLGA-Cyanine 5 endcap, which has a lactide-glycolide ratio of 50:50 and a MW of 30,000–55,000 (acid-terminated), was purchased from PolySciTech (Cat #AV034). Poly(vinyl alcohol) (PVA, 30–70 kDa) was purchased from Sigma-Aldrich (Cat #P8136). Phosphate salts were purchased from Biological Industries (Cat #11–223-1G). AM251 was purchased from Cayman Chemical Company (Cat #71670).
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