sMN differentiation was performed as previously described30 (link). For general sMN culture media, neurobasal medium (Gibco) containing B27 (Gibco), N2 (Gibco), and 2 mM L-glutamine was used as a normal medium. For compound testing, neurobasal medium with N2 was used as a conditioned medium using caffeic acid (Sigma, C0625), R-Deprenyl hydrochloride (Sigma, M003), Ajamaline (MP Biomedicals, 4360-12-7), Creatine (Sigma, 1150320) and ISP-1 (Sigma, M1177), BW755C (Tocris, 105910), Nordihydroguaiaretic acid (Sigma, 74540), Apigenin (Fisher Scientific, 50908414). For Arachidonic acid testing, Arachidonic acid (Cayman, 506-32-1) was treated in normal media. For mitomycin C treatment, 1 μg/ml of mitomycin C was treated in differentiating oMN or sMN cells for 1 hr. at D17 and analyzed after 2 days (D19) by FACS. For fold change value, non-treated % of GFP+ were considered as a control and fold change values were normalized upon % of GFP expression of non-treated cells by FACS.
Arachidonic acid
Manufactured by Cayman Chemical
Sourced in United States, Germany, Canada
Arachidonic acid is a polyunsaturated fatty acid commonly used in laboratory research. It is a key component of cell membranes and plays a role in various physiological processes.
Automatically generated - may contain errors
Lab products found in correlation
Linoleic acid, by Cayman Chemical (10 mentions)
Eicosapentaenoic acid, by Cayman Chemical (8 mentions)
Docosahexaenoic acid, by Cayman Chemical (7 mentions)
20 hete, by Cayman Chemical (5 mentions)
Oleic acid, by Cayman Chemical (5 mentions)
Calcium ionophore a23187, by Merck Group (5 mentions)
U46619, by Cayman Chemical (5 mentions)
α linolenic acid, by Cayman Chemical (4 mentions)
Dihomo γ linolenic acid, by Cayman Chemical (4 mentions)
Hanks balanced salt solution (hbss), by Lonza (4 mentions)
Acetonitrile, by Thermo Fisher Scientific (4 mentions)
Methanol, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Pgf2α, by Cayman Chemical (3 mentions)
8 iso pgf2α, by Cayman Chemical (3 mentions)
Palmitic acid, by Cayman Chemical (3 mentions)
Stearic acid, by Cayman Chemical (3 mentions)
Indomethacin, by Merck Group (3 mentions)
Urb597, by Cayman Chemical (3 mentions)
Docosahexaenoic acid dha, by Cayman Chemical (3 mentions)
95 protocols using arachidonic acid
1
sMN Differentiation and Compound Testing
2
sMN Differentiation and Compound Testing
3
Extraction and Evaluation of Bioactive Compounds from Shokyo and Kankyo
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Powders of shokyo and kankyo were provided by Tsumura & Co. 3-D HPLC profiles of shokyo and kankyo were shown in Fig. S1 . These powders were suspended in Dulbecco’s modified Eagle’s medium (D-MEM, Wako, Osaka, Japan) containing 10% heat-inactivated fetal calf serum, 100 units/ml of penicillin, and 100 mg/ml of streptomycin (culture medium), and were rotated at 4 °C overnight. Then, the suspensions were centrifuged and the supernatants were filtered through a 0.45 µm pore membrane. Lipopolysaccharide (LPS) from Porphyromonas gingivalis 381 was provided by Professor Nobuhiro Hanada (School of Dental Medicine, Tsurumi University, Japan). Arachidonic acid and 6-shogaol were purchased from Cayman Chemical (Ann Arbor, MI, USA). NF-κB inhibitor, BAY 11-7082, was purchased from Wako. Mitogen-activated protein kinase kinase (MAPKK/MEK) inhibitor, PD98059, were purchased from Sigma (St. Louis, MO). Other reagents were purchased from Nacalai Tesque.
The mouse macrophage cell line RAW264.7 (RIKEN BioResource Research Center, Tsukuba, Japan) was cultured in culture medium at 37 °C in a humidified atmosphere of 5% CO2.
The mouse macrophage cell line RAW264.7 (RIKEN BioResource Research Center, Tsukuba, Japan) was cultured in culture medium at 37 °C in a humidified atmosphere of 5% CO2.
4
Fatty Acid and RNAi Regulation Study
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DHA, arachidonic acid (AA; 20:4n-6), and linoleic acid (LA; 18:2n-6) were obtained from Cayman Chemicals and prepared as 100 mM stocks in ethanol. ON-TARGETplus siRNAs were purchased from Dharmacon. RNAiMAX was purchased from Invitrogen. The 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), collagenase (C6885), and type B bovine gelatin were purchased from Sigma-Aldrich (St. Louis, MO). GW501516 (GW1516) was purchased from R&D Systems (Minneapolis, MN). Dispase I was purchased from Wako Pure Chemicals and complete protease inhibitor cocktail was from Roche Applied Science.
5
Fatty Acid Effects on E. coli Growth
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Escherichia coli MG1655, purchased from ATCC, was used for all experiments. Overnight cultures grown in Luria broth were pelleted, washed with PBS, and transferred to M9 minimal medium [0.4% glucose supplemented with 150 mM NaCl] for initiation of most experiments. All experiments were performed at 37 °C. Fatty acids used in this study were purchased from Cayman Chemicals [linoleic acid (18:2), α-linolenic acid (18:3α), γ-linolenic acid (18:3γ), dihomo-γ-linolenic acid (20:3), arachidonic acid (20:4), eicosapentaenoic acid (20:5), and docosahexaenoic acid (22:6)].
6
Prostaglandin Synthase Activity Assay
7
Evaluating 5-LO Inhibition in Transfected HEK293 Cells
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HEK293 cells were stably co-transfected with a pcDNA3.1 vector expressing 5-LO and a pBUDCE4.1 vector expressing 5-LO activating protein (FLAP) as previously reported [29 (link)]. This represents a controlled model for the evaluation of 5-LO inhibition in a cellular system. For cell stimulation of 5-LO products, transfected HEK293 cells were collected following trypsinization, washed and the cell pellet was resuspended in Hank’s balanced salt solution (HBSS) (Lonza, Walkerville, MD) containing 1.6 mM CaCl2 at a concentration of 5 × 105 cells/mL. Cells were pre-incubated with each compound dissolved in DMSO at the indicated concentration for 5 min at 37 °C. Cells were then stimulated for 15 min at 37 °C with the addition of 10 µM calcium ionophore A23187 (Sigma-Aldrich, Oakville, ON, Canada) and 10 µM arachidonic acid (Cayman Chemical, Ann Arbor, MI). Stimulation was stopped and processed for RP-HPLC analysis as described previously [29 (link),36 (link)].
8
Lipidomics Sample Preparation Protocol
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AminoxyTMT sixplex Label Reagent Set was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Phospholipid mixtures were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Lipid mediator standards, 12-HETE, 20-HETE, TXB2, PGE2, arachidonic acid, tetranor-PGDM, and 20-carboxy-LTB4 were obtained from Cayman Chemical (Ann Arbor, MI, USA). LPA (16:0), LPA (18:1), and PA (36:2) were obtained from Avanti Polar Lipids (Alabaster, AL, USA). DMTMM and 4-methylmorpholine (NMM) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) was obtained from Fuji Film Wako Pure Chemical Co. (Osaka, Japan). Special grade reagents of ammonium bicarbonate, sodium chloride and 1-buthanol, and HPLC grade solvents of methanol, isopropanol and acetonitrile were purchased from Wako Pure Chemical Co. The authentic lipid standard stock solutions were 12-HETE (0.6 μg/mL), 20-HETE (0.6 μg/mL), TXB2 (0.6 μg/mL), PGE2 (0.6 μg/mL), AA (0.6 μg/mL), tetranor-PGDM (0.6 μg/mL), 20-carboxy-LTB4 (0.6 μg/mL), LPA(16:0) (10 μM), LPA (18:1) (10 μM), PA (36:2) (5 μg/mL).
9
Assessing Eicosanoid Signaling Pathways
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19(S)-HETE and all related HETEs, cicaprost, arachidonic acid, PGE2, PGD2, cloprostenol, U46619, PGD2, Cay10441, were from Cayman Chemicals. FR122047 and NS398 were from Tocris. Forskolin, thrombin and sodium nitroprusside (SNP) were from Sigma-Aldrich and indomethacin from Alfa Aesar. [3H]-Iloprost (20 Ci/mmol) was from American Radiolabeled Chemicals.
10
Quantification of 5-LO Products in Transfected HEK293 Cells
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Transfected HEK293 cells were collected and resuspended in Hank's balanced salt solution (HBSS, Lonza) containing 1.6 mmol/L CaCl2 at a concentration of 5 × 105 cells/mL, and were preincubated with each compound at concentration of 1 µmol/L for 5 minutes at 37°C. Cells were then stimulated for 15 minutes at 37°C with the addition of 10 µmol/L calcium ionophore A23187 (Sigma‐Aldrich) and 10 µmol/L arachidonic acid (Cayman Chemical).28 Stimulations were stopped by adding 0.5 volume of cold CH3OH:CH3CN (1:1) containing 100 ng/mL of prostaglandin B2 (PGB2) as internal standard. Samples were stored at −20°C and analyzed for 5‐LO products using reversed‐phase high‐performance liquid chromatography (RP‐HPLC) as described previously.32
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