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2 ag d8

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2-AG-d8 is a deuterated form of 2-arachidonoylglycerol (2-AG), a naturally occurring endocannabinoid found in the body. It is used as an internal standard in the analysis of 2-AG levels in biological samples.

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Lab products found in correlation

Pea d4, by Cayman Chemical (9 mentions) Aea d8, by Cayman Chemical (8 mentions) Oea d2, by Cayman Chemical (7 mentions) Dhea d4, by Cayman Chemical (6 mentions) Epea d4, by Cayman Chemical (6 mentions) Aea d4, by Cayman Chemical (3 mentions) 2 arachidonoylglycerol 2 ag, by Cayman Chemical (3 mentions) Sea d3, by Cayman Chemical (2 mentions) Oea d4, by Cayman Chemical (2 mentions) Palmitoylethanolamide, by Cayman Chemical (2 mentions) Oleoylethanolamide, by Cayman Chemical (2 mentions) N 6 arachidonoylethanolamide, by Cayman Chemical (2 mentions) 2 palmitoylglycerol 2 pg, by Cayman Chemical (2 mentions) Deuterated aea d4, by Cayman Chemical (2 mentions) N 3 docosahexaenoylethanolamide, by Cayman Chemical (2 mentions) N 3 eicosapentaenoylethanolamide, by Cayman Chemical (2 mentions) 2 palmitoylglycerol, by Cayman Chemical (2 mentions) Revertaid h minus first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions) Acetonitrile, by Merck Group (2 mentions) Rimonabant, by Merck Group (1 mentions)

Spelling variants of this product

2-arachidonoyl glycerol-d8 (2-AG-d8); (2 citations)

17 protocols using 2 ag d8

1

Preparation of Cannabinoid Compounds

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Lactic acid was purchased from Sigma Chemical Co. (St. Louis, MO, USA). AEA, AEA-d8, 2-AG, and 2-AG-d8 were purchased from Cayman Chemical (Ann Arbor, MI, USA). URB597 (cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester) (Fegley et al., 2005 (link)), rimonabant, and SR144528 (N-{(1S)-endo-1,3,3-trimethyl bicyclo[2.2.1] heptan-2yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide) (Griffin et al., 1999 (link)) were provided by the National Institute on Drug Abuse Drug Supply Program (Bethesda, MD, USA). Lactic acid was prepared in sterile water. URB597 was prepared in a vehicle consisting of 1% carboxymethylcellulose (Sigma), 1% Tween 80 (Sigma), 2% dimethyl sulfoxide (Sigma), and 96% sterile saline. rimonabant and SR144528 were prepared in a vehicle consisting of 5% ethanol, 5% cremophor (Sigma), and 90% sterile saline. All solutions were injected i.p. in a volume of 1 ml/kg except for URB597, which was injected i.p. in a volume of 2 ml/kg.
Kwilasz A.J., Abdullah R.A., Poklis J.L., Lichtman A.H, & Negus S.S. (2014). Effects of the Fatty Acid Amide Hydrolase (FAAH) Inhibitor URB597 on Pain-Stimulated and Pain-Depressed Behavior in Rats. Behavioural pharmacology, 25(2), 119-129.
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2

Lipid Extraction and Quantification from Mouse Brain and Serum

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Lipids were extracted from mouse brain or serum essentially as described in (Wang et al. 2003 (link)) with the following modifications: Frozen mouse brain (100–200 mg tissue) was homogenized in 1.0 mL of ice-cold homogenization buffer containing 2000 pg each of the following deuterated standards (AEA-d4, OEA-d2, PEA-d4, DHEA-d4, EPEA-d4, and 2-AG-d8; Cayman Chemical, Ann Arbor, MI). Similar amounts of the respective deuterated standards were added to an aliquot (50 μL) of the serum prior to lipid extraction as described above. The final lipid residue was reconstituted in 100 μL of ice-cold methanol, purged with nitrogen, and stored at −80°C until analysis by liquid chromatography-mass spectrometry (LC-MS).
Martin G.G., Chung S., Landrock D., Landrock K.K., Huang H., Dangott L.J., Peng X., Kaczocha M., Seeger D.R., Murphy E.J., Golovko M.Y., Kier A.B, & Schroeder F. (2016). FABP-1 GENE ABLATION IMPACTS BRAIN ENDOCANNABINOID SYSTEM IN MALE MICE. Journal of neurochemistry, 138(3), 407-422.
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3

Quantification of Endocannabinoids and Lipid Mediators

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Radioactive arachidonoyl ethanolamide[1-3H] ([3H]-AEA) was obtained from American Radiolabeled Chemicals, Inc (St Louis, MO, USA). (R)(-)-Flurbiprofen and (S)(+)-Flurbiprofen were purchased from Santa Cruz Biotechnology Inc. (Dallas, Texas, USA). Ovine COX-1 (cat. no. 60100), human recombinant COX-2 (cat. no. 60122), COX-2 polyclonal antibody (rabbit anti-mouse, cat #: 160106), arachidonic acid, 2-AG, AEA and URB597 (cyclohexylcarbamic acid 3′-carbamoylbiphenyl-3-yl ester) were purchased from the Cayman Chemical Co. (Ann Arbor, MI, USA). Substrates were dissolved in ethanol or DMSO as appropriate. Polyclonal goat anti-rabbit immunoglobulin/HRP was obtained from Dako (Glostrup, Denmark). Protease inhibitor cocktail set III was obtained from Merck Millipore (Darmstadt, Germany). For the lipid quantification experiments, the following native and deuterated standards were purchased from the Cayman Chemical Co.: AEA, 2-AG, PEA, OEA, DEA, LEA, SEA, 2-LG, AEA-d8, 2-AG-d8, PEA-d4, SEA-d3, OEA-d4, PGF2α, PGE2, TXB2, PGD2, 12(13)-EpOME, 9(10)-DiHOME, 12(13)-DiHOME, 11-HETE, 12-HETE, 15-HETE, 9-HODE, 13-HODE, 12-HEPE, 12-[[(cyclohexylamino)carbonyl]amino]-dodecanoic acid (CUDA), 12(13)-DiHOME-d4, 12(13)-EpOME-d4, 9-HODE-d4, PGE2-d4 and PGD2-d4. 9,10,13-TriHOME and 9,12,13-TriHOME were obtained from Larodan (Sweden, Malmö). For list of lipid abbreviations, see S1 Table.
Gouveia-Figueira S., Karlsson J., Deplano A., Hashemian S., Svensson M., Fredriksson Sundbom M., Congiu C., Onnis V, & Fowler C.J. (2015). Characterisation of (R)-2-(2-Fluorobiphenyl-4-yl)-N-(3-Methylpyridin-2-yl)Propanamide as a Dual Fatty Acid Amide Hydrolase: Cyclooxygenase Inhibitor. PLoS ONE, 10(9), e0139212.
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4

Quantifying Endocannabinoid Signaling Molecules

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Dulbecco’s modified Eagle’s medium, foetal calf serum and penicillin/streptomycin were from Corning (Corning, NY, USA). Bovine serum albumin (BSA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Sphingosine-1-phosphate (S1P, cod.62570) and methanandamide (N-(2-hydroxy-1R-methylethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide, cod.157182-49-5) were from Cayman Chemicals (Ann Arbor, MI, USA); N-arachidonoyl-ethanolamine (anandamide (AEA), cod. A0580) and 2-arachidonoil-glycerol (2-AG, cod. A8973) were from Sigma-Aldrich (St. Louis, MO, USA). AEA-d8 and 2-AG-d8 were purchased from Cayman Chemicals (Ann Arbor, MI, USA) and were used as internal standards. The selective antagonists of CB1 SR141716A (SR1, cod. SML0800), of CB2 SR144528 (SR2, cod. SML1899) and of GPR55 ML193 trifluoroacetate (ML193, cod. SML1340) were from Sigma-Aldrich (St. Louis, MO, USA). The TRPV1 selective antagonist 5′-iodoresiniferatoxin (I-RTX, cod. 1362) was from TOCRIS (Bristol, UK). For molecular biology studies RevertAid H Minus First Strand cDNA Synthesis Kit, from Thermo Scientific (Waltham, MA, USA), and SensiFASTTM SYBR Lo-ROX kit, from Bioline (London, UK) were used.
Standoli S., Pecchioli S., Tortolani D., Di Meo C., Fanti F., Sergi M., Bacci M., Seidita I., Bernacchioni C., Donati C., Bruni P., Maccarrone M., Rapino C, & Cencetti F. (2022). The TRPV1 Receptor Is Up-Regulated by Sphingosine 1-Phosphate and Is Implicated in the Anandamide-Dependent Regulation of Mitochondrial Activity in C2C12 Myoblasts. International Journal of Molecular Sciences, 23(19), 11103.
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5

Quantitative Lipid Profiling of Mouse Liver

Cited 3 times
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Lipids were extracted from mouse livers as described earlier [11 (link), 28 ]. Briefly, frozen mouse liver (100–200 mg wet weight) was homogenized in 1.0 mL of ice-cold homogenization buffer containing each of the deuterated standards AEA-d4, OEA-d2, PEA-d4, DHEA-d4, EPEA-d4, and 2-AG-d8 such that the final amount of each standard was 2000pg. The final lipid residue was redissolved in 100 μL of ice-cold methanol, purged with N2, and stored at −80°C until liquid chromatography-mass spectrometry (LC-MS) analysis below.
LC/MS analysis of liver NAE and 2-MG was performed as described earlier by our lab [30 (link), 31 (link)]. Briefly, identification and quantitation of individual NAE or 2-MG was accomplished through use of: i) Addition of internal standards to each aliquot of liver homogenate including deuterated AEA-d4, OEA-d2, PEA-d4, DHEA-d4, EPEA-d4, and 2-AG-d8 from Cayman Chemical (Ann Arbor, MI); ii) Comparison with external standard curves of n-6 arachidonoylethanolamide (AEA), oleoylethanolamide (OEA), palmitoylethanolamide (PEA), n-3 docosahexaenoylethanolamide (DHEA), n-3 eicosapentaenoylethanolamide (EPEA), 2-arachidonoylglycerol (2-AG), 2-oleoylglycerol (2-OG), and 2-palmitoylglycerol (2-PG) from Cayman Chemical (Ann Arbor, MI). All reagents and solvents used for extraction and LC/MS were of the highest commercial grade available.
Martin G.G., Seeger D.R., McIntosh A.L., Chung S., Milligan S., Landrock D., Dangott L.J., Golovko M.Y., Murphy E.J., Kier A.B, & Schroeder F. (2018). Scp-2/Scp-x Ablation in Fabp1 Null Mice Differentially Impacts Hepatic Endocannabinoid Level Depending on Dietary Fat. Archives of biochemistry and biophysics, 650, 93-102.
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6

Lipid Extraction from Skin Samples

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Skin samples were thawed at 4 °C in ice. After weighing, 500 µL of phosphate buffer 0.1 mM pH 5.6 and 2 µL ISTD spiking solution containing AEA-d8, PEA-d4, OEA-d2 and 2AG-d8 (Cayman Chemical) were added to all samples. Chloroform:MeOH (2:1) 500 µL was added, and the samples were then vortexed vigorously for 2 cycles of 5 s at 5,000 rpm using a bead beater and centrifuged at 20,000 × g for 10 min at 4 °C, after which the organic layer was removed. This procedure was repeated three times, and all the organic phases were pooled. Then, the organic phase was evaporated in a CentriVap concentrator at 50 °C until dryness and reconstituted in 100 µL of acetonitrile. The supernatant was then transferred to HPLC vials to be injected (5 µL) into an LC–MS/MS device.
Correia-Sá I.B., Carvalho C.M., Serrão P.V., Loureiro A.I., Fernandes-Lopes C., Marques M, & Vieira-Coelho M.A. (2020). A new role for anandamide: defective link between the systemic and skin endocannabinoid systems in hypertrophic human wound healing. Scientific Reports, 10, 11134.
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7

Quantification of Endocannabinoid Metabolites

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Unlabeled AEA (n-6 arachidonoylethanolamide), OEA (oleoylethanolamide), PEA (palmitoylethanolamide), DHEA (n-3 docosahexaenoylethanolamide), EPEA (n-3 eicosapentaenoylethanolamide), 2-AG (2-arachidonoylglycerol), 2-OG (2-oleoylglycerol), and 2-PG (2-palmitoylglycerol) were purchased from Cayman Chemical (Ann Arbor, MI). Deuterated AEA-d4, OEA-d2, PEA-d4, DHEA-d4, EPEA-d4, and 2-AG-d8 were also from Cayman Chemical (Ann Arbor, MI).
Huang H., McIntosh A.L., Martin G.G., Landrock D., Chung S., Landrock K.K., Dangott L.J., Li S., Kier A.B, & Schroeder F. (2016). FABP1: A NOVEL HEPATIC ENDOCANNABINOID AND CANNABINOID BINDING PROTEIN. Biochemistry, 55(37), 5243-5255.
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8

Inhibition of ABHD6 and ABPP Assay

Cited 1 time
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KT182, an ABHD6 inhibitor, and HT-01, the activity-based protein profiling (ABPP) probe specific for ABHD6, were kindly provided by Drs. Hsu and Cravatt [26 (link)]. 2-Arachidonoylglycerol [glycerol-1,2,3-3H] was from American Radiolabeled Chemicals Inc. (Saint Louis, MO). Cyclooxygenase inhibitor assay kits including COX inhibitor and recombinant COX-1 or COX-2 activity assay kit were from Cayman Chemical (Ann Arbor, MI). siRNA (FlexiTube Mm_abhd6_3 and Allstars Negative Control) and HiPerfect transfection reagent were from QIAGEN (Valencia, CA). WWL70, methyl arachidonyl fluorophosphonate (MAFP), SR141716 (SR1), SR144528 (SR2), 2-AG, 2-AG-d8, and AA were purchased from Cayman Chemical. Other reagents including lipopolysaccharide (LPS) were purchased from Sigma-Aldrich (St. Louis, MO).
Tanaka M., Moran S., Wen J., Affram K., Chen T., Symes A.J, & Zhang Y. (2017). WWL70 attenuates PGE2 production derived from 2-arachidonoylglycerol in microglia by ABHD6-independent mechanism. Journal of Neuroinflammation, 14, 7.
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9

Quantitative Lipid Profiling Methodology

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Purified unlabeled N-acylethanolamides (NAEs) and 2-monoacylglycerols (2-MGs) as well as their deuterated analogues were from Cayman Chemical (Ann Arbor, MI) as follows: n-6 arachidonoylethanolamine (AEA), oleoylethanolamide (OEA), palmitoylethanolamide (PEA), n-3 docosahexaenoylethanolamide (DHEA), n-3 eicosapentaenoylethanolamide (EPEA), 2-arachidonoylglycerol (2-AG), 2-oleoylglycerol (2-OG), 2-palmitoylglycerol (2-PG), AEA-d4, OEA-d2, PEA-d4, DHEA-d4, EPEA-d4, 2-AG-d8, and arachidonic acid-d8 (ARA-d8, 20:4n-6-d8. Cis-parinaric acid was from Invitrogen (Grand Island, NY, USA). All reagents and solvents were the highest grade commercially available.
Martin G.G., Chung S., Landrock D., Landrock K.K., Huang H., Dangott L.J., Peng X., Kaczocha M., Seeger D.R., Murphy E.J., Golovko M.Y., Kier A.B, & Schroeder F. (2016). FABP-1 GENE ABLATION IMPACTS BRAIN ENDOCANNABINOID SYSTEM IN MALE MICE. Journal of neurochemistry, 138(3), 407-422.
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10

Diacylglycerol Lipase Activity Assay

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10 μL DAG (10 mg/ml in CH3CN) was mixed with 50 μL Lyso phosphatidyl choline (LPC-SIGMA at 5 mg/ml in CHCL3), evaporated to dryness, solubilized with 30 μL CHCI3 followed by 100 μL of homogenizing buffer (0.1 M MOPS pH 7.4, 0, 25% BSA) and then subjected to ultrasound using a warming ultrasonic bath (Deltasonic) in an open tube for 15 min at 37°C, allowing slow CHCL3 evaporation and liposome generation.
DAGL activity. was measured at 37°C for 20 hours with 90 μL of 100 mM MOPS pH 7.4, 100 μL of purified fraction from brain, or equivalent quantity of recombinant ABHD11, and 10 μL DAG in LPC liposome as substrate (150 μΜ final DAG) or variable amount for enzymatic characterization.
For PMSF and RHC80267 inhibition, preincubation was carried out for 30 min at 3 mM and 1 mM, respectively, before adding DAG. RHC80267 (Sigma Aldrich) was also used. The reactions were stopped by adding chloroform supplemented with internal standard 2-AG-D8 (Cayman chemicals) for extraction. After centrifugation (5 min at 1500g), the organic layers were collected and dried under vacuum. The residues were suspended with 50 μL of H20/CH3CN mix (80:20) and analyzed by high performance liquid chromatography/mass spectrometry in "MRM-like" mode.
Escoubet J., Kenigsberg M., Derock M., Yaligara V., Bock M.D., Roche S., Massey F., de Foucauld H., Bettembourg C., Olivier A., Berthemy A., Capdevielle J., Legoux R., Perret E., Buzy A., Chardenot P., Destelle V., Leroy A., Cahours C., Teixeira S., Juvet P., Gauthier P., Leguet M., Rocheteau-Beaujouan L., Chatoux M.A., Deshayes W., Clement M., Kabiri M., Orsini C., Mikol V., Didier M, & Guillemot J.C. (2020). ABHD11, a new diacylglycerol lipase involved in weight gain regulation. PLoS ONE, 15(6), e0234780.
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