The following reagents were used in the experiments described below: docosahexaenoic acid (DHA), linoleic Acid (LA), 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) and resolvin D1 (RvD1) (Cayman chemicals, Ann Arbor, Michigan). Additional materials used included Collagenase II (Worthington Biochemical, Lakewood, NJ), DNase I (AppliChem, Ottoweg Darmstadt), RPMI 1640, and DMEM/F12 media fetal bovine serum (FBS) (Thermo Fischer Scientific, Grand Island, NY) Bovine serum albumin (BSA; Fraction V; Sigma Aldrich St Louis, MO) and radio-immunoprecipitation assay (RIPA) lysis buffer (Sigma Aldrich St Louis, MO). The protease inhibitor cocktail was purchased from Roche GmbH, Germany.
Resolvin d1 rvd1
Manufactured by Cayman Chemical
Sourced in United States
Resolvin D1 (RvD1) is a lipid mediator derived from the omega-3 fatty acid docosahexaenoic acid (DHA). RvD1 is involved in the resolution of inflammation, a natural process that helps restore tissue homeostasis. As a laboratory product, RvD1 is used for research purposes to study its role in various biological processes.
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Lab products found in correlation
Radio immunoprecipitation assay ripa lysis buffer, by Merck Group (1 mentions)
Dnase 1, by AppliChem (1 mentions)
Collagenase 2, by Worthington (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Bovine serum albumin fraction 5, by Merck Group (1 mentions)
Formic acid, by Thermo Fisher Scientific (1 mentions)
Methanol, by Thermo Fisher Scientific (1 mentions)
Acetonitrile, by Thermo Fisher Scientific (1 mentions)
Glutamine, by Merck Group (1 mentions)
Alanine, by Merck Group (1 mentions)
Urea, by Merck Group (1 mentions)
Taurine, by Merck Group (1 mentions)
Ethanolamine, by Merck Group (1 mentions)
Glycine, by Merck Group (1 mentions)
Citrulline, by Merck Group (1 mentions)
Phenylalanine, by Merck Group (1 mentions)
Glutamic acid, by Merck Group (1 mentions)
Threonine, by Merck Group (1 mentions)
Proline, by Merck Group (1 mentions)
Leucine, by Merck Group (1 mentions)
7 protocols using resolvin d1 rvd1
1
Lipid Mediators in Inflammatory Response
2
Untargeted Metabolomics Mass Spectrometry
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All solvents for untargeted mass spectrometry were LC-MS grade. Acetonitrile, methanol, and formic acid were purchased from Fisher Scientific (Fairlawn, New Jersey); Water was purchased from Honeywell (Muskegon Michigan); 1-methylhistidine, 3-methylhistidine, α-amino-n-butyric acid, alanine, anserine, arginine, asparagine, aspartic acid, β-aminoisobutryic acid, β-alanine, carnosine, citrulline, creatinine, cystathionine, cysteine, ethanolamine, γ-aminobutyric acid, glutamic acid, glutamine, glycine, histidine, homocystine, hydroxylysine, hydroxyproline, isoleucine, L-aminoadipic acid, L-cystine, leucine, lysine, proline, methionine, ornithine, phenylalanine, phosphoserine, phosphoethanolamine, sarcosine, serine, taurine, threonine, tryptophan, tyrosine, urea, and valine were purchased from Sigma Aldrich (St. Louis, Missouri); 10(S),17(S)-DiHDoHE (Protectin DX), 11β-PGF2α, 14(S)-HDHA, 15R-PGF2α, 17(S)-HDHA, 8-iso-15R-PGF2α, 8-iso-PGF2α, Lipoxin A4 (LXA4), LTB4, LTC4, LTD4, LTE4, PGE2, PGF2α, Resolvin D1 (RVD1), and Resolvin D2 (RVD2) were purchased from Cayman Chemical (Ann Arbor, Michigan).
All standards and deuterated internal standards used for LC-MS/MS analysis of arachidonic acid, docosahexaenoic acid derived lipid mediators were purchased from Cayman Chemical (Ann Arbor, Michigan, USA). All HPLC solvents and extraction solvents were HPLC grade or better.
All standards and deuterated internal standards used for LC-MS/MS analysis of arachidonic acid, docosahexaenoic acid derived lipid mediators were purchased from Cayman Chemical (Ann Arbor, Michigan, USA). All HPLC solvents and extraction solvents were HPLC grade or better.
3
Molecular Signaling Pathway Analysis
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Dulbecco’s modified Eagle’s medium (DMEM) and Lipofectamine 2000 were obtained from Invitrogen (Carlsbad, CA, USA). DHA, EPA, PUFA analytical standards and Matrigel were obtained from Sigma-Aldrich (St. Louis, MO, USA). The protein assay was from Bio-Rad (Hercules, CA, USA). Water-soluble tetrazolium (WST) reagent was from Dojindo Laboratories (Kumamoto, Japan). Electrochemiluminescence (ECL) reagents were obtained from Amersham Biosciences (Piscataway, NJ, USA). The dual-luciferase reporter assay system was obtained from Promega Corporation (Madison, WI, USA). PrimeScript RT Reagent Kit was obtained from TAKARA Bio Inc. (#RR037A, Shiga, Japan). SYBR Green Master was from Roche Diagnostics (#04913914001, Indianapolis, IN, USA). Resolvin D1 (RvD1) was obtained from Cayman Chemical Co. (#10012554, Ann Arbor, Michigan, USA). The following were commercially obtained antibodies: the anti-Akt (#9272), anti-phospho-Akt (Ser473, #4060), the anti-Erk1/2 (#4696), and anti-phospho-Erk1/2 (Thr202/Tyr204, #8544) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA); the anti-FOXC1 antibody (#115201) was obtained from Abcam plc (Cambridge, UK); the anti-GAPDH antibody was obtained from Bioworld Technology (Atlanta, Georgia30,305, USA). EnVision+single reagents (Mouse, Rabbit) were from DAKO (K4000, K4002, Glostrup, Denmark).
4
Curcuminoids and Resolvin D1 Emulsion
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Curcuminoids (prepared from Curcuma longa L root by extraction with acetone as granulated powder by Naturex, Avignone, France) were prepared by sonication as an emulsion in SmartfishR by the Smartfish company. Resolvin D1 (RvD1) (Cayman Chemical company, Ann Arbor, MI) was emulsified in SmartfishR.
5
Liquid Chromatography-Mass Spectrometry for Lipid Mediator Analysis
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Samples of the LV and spleen for liquid chromatography–tandem mass spectrometry analysis were obtained using solid phase extraction columns, as described.12 To observe and measure the levels of lipid mediators in the LVI, an LC‐tandem MS/MS system, QTrap 5500 (ABSciex), equipped with an Agilent HP1100 binary pump, was used. The Aligent Eclipse Plus C18 column (50 mm×4.6 mm×1.8 μm or 100 mm×4.6 mm×1.8 μm) with a gradient of methanol/double‐distilled H2O/acetic acid (60:40:0.001 to 100:0:0.01) was used at a flow rate of 0.5 mL/min. If a standard for a product was not available, a product with similar chromatographic behaviors was used for a calibration curve. Deuterium‐labeled internal standards included lipoxin A4, resolvin D1 (RvD1), Prostaglandin E2 (PGE2), leukotriene B4, and others, obtained from Cayman Chemical Company (Ann Arbor, MI) and used to calculate recoveries. The peak area of each Multiple Reaction Monitoring (MRM) transition and linear calibration curves were used for the quantification of each mediator27 A minimum of 6 characteristics and diagnostic ions were used for identification of all SPMs, in accordance with published criteria of biologic and synthetic mediators.27 , 28
6
Quantification of Plasma Biomarkers
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All immunoassay kits were measured in plasma. Myeloperoxidase (MPO) and xanthine oxidase (XOD) (Cusabio Technology LLC®, Houston, TX, USA), irisin (Cell Biolabs®, San Jose, CA, USA), and resolvin D1 (RvD1) (Cayman Chemical®, Ann Arbor, MI, USA) were determined using ELISA kits following the manufacturers’ instructions. Cytokeratin 18 (CK-18) concentration was determined using the M30 Apoptoense® ELISA kit following the guidelines for use (PEVIVA®, in USA, Canada, and Japan). Tumor necrosis factor alpha (TNFα) and Interleukin-6 (IL-6) levels were determined in plasma using Human Custom ProcartaPlexTM (Invitrogen by Thermo Fisher Scientific, Bender MedSystems GmbH, Vienna, Austria) following the manufacturers’ instructions.
7
Resolvin D1 Modulates Inflammatory Response in Periodontal Ligament Cells
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Lipopolysaccharide (LPS) was used to establish an pro-inflammatory phenotype for PDLCs. PDLCs were seeded into 6-well plates at a density of 7×10 4 cells per well and were incubated with 0.01, 0.1 or 1 μg/mL LPS for 3 days. An enzyme-linked immunosorbent assay (ELISA) was used to measure the level of IL-1β (Jianglai, Shanghai, China).
Based on the results of MTT and ELISA tests, LPS treatment with a dose of 0.1 μg/ml was found to show no toxicity on cell proliferation and can significantly stimulate IL-1β expression, therefore it was chosen in the later experiments. PDLCs were seeded into 6-well plates at a density of 7×10 4 cells per well under three different conditions (Control, LPS, LPS+RvD1) under normoxic or hypoxic conditions.
Resolvin D1 (RvD1, Cayman Chemical, Ann Arbor, Michigan) at 100 ng/ml was used according to previous studies (17, (link)32) (link). The normoxic groups were cultured in an incubator with 5% CO 2 at 37 °C. The hypoxic groups were placed in the hypoxic workstation (Maworde, Jiangsu, China) in which the pressure of oxygen (pO 2 ) is 7.14 mmHg [1.0% (vol/vol) O 2 ], the pressure of carbon dioxide is 35 mmHg [5% (vol/vol) CO 2 ] and the temperature is 37 °C. After 3 days culturing, cells and supernatants were collected separately for different measurements.
Based on the results of MTT and ELISA tests, LPS treatment with a dose of 0.1 μg/ml was found to show no toxicity on cell proliferation and can significantly stimulate IL-1β expression, therefore it was chosen in the later experiments. PDLCs were seeded into 6-well plates at a density of 7×10 4 cells per well under three different conditions (Control, LPS, LPS+RvD1) under normoxic or hypoxic conditions.
Resolvin D1 (RvD1, Cayman Chemical, Ann Arbor, Michigan) at 100 ng/ml was used according to previous studies (17, (link)32) (link). The normoxic groups were cultured in an incubator with 5% CO 2 at 37 °C. The hypoxic groups were placed in the hypoxic workstation (Maworde, Jiangsu, China) in which the pressure of oxygen (pO 2 ) is 7.14 mmHg [1.0% (vol/vol) O 2 ], the pressure of carbon dioxide is 35 mmHg [5% (vol/vol) CO 2 ] and the temperature is 37 °C. After 3 days culturing, cells and supernatants were collected separately for different measurements.
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