Leukotriene b4

Methanol, acetonitrile (ACN), ethyl acetate, water, formic acid (FA) were acquired from Merck Millipore (Warsaw, Poland). Standards of Thromboxane B2, Leukotriene B4, Prostaglandin D2, Prostaglandin E2, 6-keto Prostaglandin F1α, Prostaglandin F2α, 15-deoxy-Δ12,14-Prostaglandin J2, 13,14-dihydro Prostaglandin E1, and their isotope-labeled standards were procured from Cayman Chemical Company (Ann Arbor, MI, USA).

Mouse monoclonal antibody for α-tubulin, C23, NF-κB p65 and rabbit polyclonal antibody for IgG, IKKα/β, IκBα, NF-κB p50, NF-κB p65 and goat anti-mouse or anti-rabbit secondary antibody conjugated with horseradish peroxidase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse monoclonal antibody for phosphor-IκBα and rabbit monoclonal antibody for phosphor-IKKα/β were from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti-5-LOX antibody was from Novus (Littleton, CO, USA). 5-LOX inhibitors, including MK-886, Nordihydroguaiaretic acid (NDGA); leukotriene B4 and leukotriene B4 receptor antagonist LY29311 were from Cayman Chemical Company (Ann Arbor, MI, USA). Collagenase and 4′,6-diamidino-2-phenylindole (DAPI) were from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human TNF-α and enhanced chemiluminescent HRP substrate (ECL) were from Millipore (Bedford, MA, USA). We purchased RPMI-1640 medium, trypsin and anti-rabbit secondary antibody conjugated with Alexa Fluor 488 from Invitrogen (Carlsbad, CA, USA) and fetal bovine serum (FBS) from Biological Industries (Kibbutz Beit Haemek, Israel). Tri-zol was from MDBIO (Taipei, Taiwan). MMLV Reverse Transcriptase kit was from Promega (Madison, WI, USA). Taqman PCR Master Mix and qPCR probes were from Applied Biosystems/Invitrogen (Foster city, CA, USA).

Prostaglandin E₂ (PGE₂), prostaglandin D₂ (PGD₂), leukotriene B₄ (LTB₄), 6-trans-leukotriene B₄ (t-LTB₄), 6-trans-12-epi-leukotriene B₄ (trans-epi-LTB₄), 12-epi-leukotriene B₄ (epi-LTB₄), 20-hydroxyleukotriene B₄ (20-OH-LTB₄), thromboxane B₂ (TxB₂), 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE), 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), 5(S)-hydroxyeicosatetraenoic acid (5(S)-HETE), prostaglandin B₁ (PGB₁), prostaglandin F_2α (PGF_2α), and zileuton were purchased from Cayman Chemicals (Ann Abor, USA). MS-grade methanol (MeOH), isopropanol (IPA), and acetonitrile (ACN) were from VWR (Darmstadt, Germany), ammonium hydroxide from Honeywell (Charlotte, USA), and formic acid, diclofenac, and acetylsalicylic acid (ASS) from Merck KGaA (Darmstadt, Germany). Lipopolysaccharide (LPS), Histopaque-1077^®, n-hexane, and methyl formate were from Merck (Darmstadt, Germany). L-glutamine and CaCl₂ were purchased from Carl Roth (Karlsruhe, Germany).

LC-MS/MS-based lipid mediator and oxylipin profiling were carried out as described elsewhere [4 (link)]. A QTrap 6500 mass spectrometer (Sciex) was used, coupled to a Shimadzu Nexera LC30-system including auto-sampler and column oven (Shimadzu). The column was a Kinetex C18 50 × 2.1 mm, 1.7 μm, protected with a C8 pre-column (Phenomenex). LC-MS/MS peaks were integrated with manual supervision, and the areas were corrected to corresponding IS using MultiQuant™ 2.1 (Sciex). For quantitation, the multiple reaction monitoring (MRM) transitions and collision energies (CE) were used together with calibration lines for quantification. Calibration lines were constructed using 15-HETE, 17R-HDHA, leukotriene B₄, prostaglandin E₂, arachidonic acid, docosahexaenoic acid, and eicosapentaenoic (Cayman Chemicals)

For the assessment of the cellular status of enzymes such as glutathione (ab65322) cathepsin B (ab65300), cathepsin D (ab65302), myeloperoxidase (MPO) (ab105136), commercial enzyme assay kits were used and the assays were performed as per the manufacturer’s protocol (Abcam Inc, Massachusetts, USA). To elucidate the inflammatory responses, the marker enzymes such as cyclooxygenase (COX-2, (ab204699), lipooxygenase (5- LOX, ab241038), sPLA2 and leukotriene B4 (Cayman Chemicals, USA) were estimated in the cells using commercial assay kits (Wuhan Fine Biotech Co., Ltd., Hubei, China).

Chemical reagents and HPLC grade organic solvents were purchased from Sigma Aldrich (St. Louis, MO, USA). These include HPLC grade Methanol, Chloroform, Acetonitrile, Isopropanol, Butylated Hydroxytoluene, Ammonium Formate, and Formic Acid. Synthetic lipid standards 21:0–22:6 PC was purchased from Avanti Polar Lipids (Alabaster, AL, USA). Oxylipin standards 9,10-DIHOME, 20-HETE, 9-HETE, 8,9-DHET, 14,15-DHET, 5-HETE, 12-HETE, 11,12-DHET, 5,6-DHET, 13-KODE, 13-HpODE, 9-HpODE, 9-HODE, 9-HoTre, 13-HoTre, 14(15)-EET, 8(9)-EET, 11(12)-EET, 12(13)-EPOME, 13-HODE, Prostaglandin E2, Prostaglandin E2 Ethanolamide, Prostaglandin E2 Glycerol, ARA-Ethanolamide (AEA), 2-AG, Oxy-AEA, Oleamide, Docosanoyl Ethanolamide, Docosahexaneoyl Ethanolamide (DHEA), LinOleoyl Ethanolamide (LEA), dihomo-gamma Linolenoyl Ethanolamide, a-Linolenoyl Ethanolamide (ALEA), Resolvin D3, Resolvin D1, Resolvin D2, Resolvin E1, Thromboxane B2, Leukotriene B4, Prostaglandin F2a, 8-Isoprostane, Oleoyl Ethanolamide (OEA), Palmitoyl Ethanolamide (PEA), Stearoyl Ethanolamide (SEA) were purchased from Cayman Chemical (Ann Arbor, MI, USA).

Prostaglandins were extracted from the kidney homogenate by adding 0.5 mL of water–ethanol (1:4 vol/vol) and 10 μL of glacial acetic acid to 25 µg of protein. The mixture was well shaken and incubated at room temperature for 5 min and centrifuged at 2500× g for 5 min. The supernatant was applied to a Sep-Pak C18 mini-column (Millipore, Billerica, MA, USA) previously equilibrated with 2 mL of 10% ethanol. The column was then washed with 1 mL of water followed by 1 mL of hexane, and prostaglandins were eluted with 1.50 mL of ethyl acetate. The samples were dried under a nitrogen stream and re-suspended in 300 μL of phosphate-buffered saline–ethanol (2:1 vol/vol) [14 (link)]. The prostaglandins were determined with ELISA kits; prostaglandin E (Item No. 514531), 6-keto prostaglandin F_1α (Item No. 515211), leukotriene B₄ (Item No. 520111), and thromboxane B₂ (Item No. 501020) were obtained from Cayman Chemical (Ann Arbor, MI, USA), and were read in a visible light micro plate reader at 492/630 nm.