Cells seeded into 48-well plates (5 x 104/well) or 12-well plates (1 x 105/well) in complete medium and grown to confluence. Cultures were rinsed with HBSS and shifted to M199+0.5% charcoal-stripped FBS/5% bovine endothelial cell growth supplement (ECGS, ThermoFisher Scientific) for experiments. Culture media were collected, cells were solubilized with 1N NaOH and samples were frozen -80°C. Specific ELISAs for 6-ketoprostaglandin F1α (6-ketoPGF1α), prostaglandin E2 (PGE2), Lipoxin A4 (LXA4), 15-epi-Lipoxin A4 (15-epi-LXA4) and Leukotriene B4 (LTB4) (Cayman Chemical, Ann Arbor, MI) were used to measure analytes following incubation with test substances. Total cell protein was measured in each well and data were expressed as ng of analyte/mg protein. All measurements were made using a Perkin Elmer Victor V3™ multichannel microplate reader. To control for possible endogenous lipid mediators in the culture media, each analyte was measured in culture media (M199+0.5% FCS/5% ECGS) that was not incubated with cells.
Leukotriene b4 ltb4
Manufactured by Cayman Chemical
Sourced in United States
Leukotriene B4 (LTB4) is a lipid mediator produced by the metabolism of arachidonic acid. It is a potent chemoattractant and activator of leukocytes, playing a role in inflammatory and immune responses.
Automatically generated - may contain errors
Lab products found in correlation
Countbright counting beads, by Thermo Fisher Scientific (3 mentions)
Recombinant mouse tnfα, by Thermo Fisher Scientific (3 mentions)
Ifn γ, by Thermo Fisher Scientific (3 mentions)
Ammonium chloride potassium ack lysing buffer, by Lonza (3 mentions)
Edta 0.5m, by Lonza (3 mentions)
Hepes 1m, by Lonza (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (3 mentions)
Fibronectin, by Merck Group (2 mentions)
Recombinant murine e selectin fc, by R&D Systems (2 mentions)
Vcam 1 fc and icam 1 fc chimera molecules, by R&D Systems (2 mentions)
Polyclonal goat anti mouse cd47, by R&D Systems (2 mentions)
Goat anti human jam a, by R&D Systems (2 mentions)
Recombinant protein g sepharose 4b, by Thermo Fisher Scientific (2 mentions)
Prolong antifade mounting agent, by Thermo Fisher Scientific (2 mentions)
Proteinase inhibitor cocktail, by Merck Group (2 mentions)
Histopaques 1077 and 1119, by Merck Group (2 mentions)
2 2 azino di 3 ethyl di thiazoline sulfonic acid, by Merck Group (2 mentions)
Duolink in situ fluorescence kit, by Merck Group (2 mentions)
Penicillin streptomycin, by Corning (2 mentions)
13 protocols using leukotriene b4 ltb4
1
Lipid Mediator Quantification in Endothelial Cells
2
Neutrophil Activation Assays Utilizing Diverse Reagents
3
Neutrophil Isolation and Stimulation Protocol
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Ammonium–Chloride–Potassium (ACK) lysing buffer, EDTA 0.5 M, and HEPES 1 M were purchased from Lonza (Walkersville, MD, USA). Hanks’ Balanced Salt Solution with Ca2+ and Mg2+ (HBSS+), and Phosphate Buffered Saline without Ca2+ and Mg2+ (PBS−) were purchased from Corning Cellgro, Mediatech Inc. (Tewksbury, MA, USA). Rhodamine 70 kDa dextran, Hoechst 33,342 and CountBright counting beads were purchased from Invitrogen (Carlsbad, CA, USA). Fetal bovine serum (FBS) was obtained from Atlanta Biologicals (Oakwood, GA, USA). Leukotriene B4 (LTB4) was purchased from Cayman Chemical (Ann Arbor, MI, USA), N-Formylmethionyl-leucyl-phenylalanine (fMLF) and Plerixafor (AMD3100 octahydrochloride hydrate) were purchased from Millipore-Sigma (St. Louis, MO, USA). CXCL-1 was obtained from R&D Systems (Minneapolis, MN, USA). Recombinant mouse TNFα and IFNγ were purchased from Peprotech (Rocky Hill, NJ, USA). A complete list of antibodies clones and concentrations used in this work is provided as Supplementary Material (Supplementary Table S2 ).
4
Arachidonic Acid and Inflammation Modulators
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Arachidonic acid (AA: 5,8,11,14-eicosatetraenoic acid), dexamethasone [DEX (11β, 16α)-9-fluoro-11,17,21-trihydroxy-16-methylpregna-1,4-diene-3], naproxen [NAP (S)-(+)-2-(6-methoxy-2-naphthyl)propionic acid], esculetin (ESC: 6-hydroxy-7-methoxycoumarin), 3-ethoxy-4-methoxyphenol (EMP) as a specific inhibitor to DSP1 [22 (link)], and PGE2 ((5Z,11α,13E,15S)-11,15-dihydroxy-9-oxoprosta-5,13-dienoic acid) were purchased from Sigma-Aldrich Korea (Seoul, Korea) and leukotriene B4 (LTB4: 5S,12R-dihydroxy-6Z,8E,10E,14Z-eicosatetraenoic acid) was purchased from Cayman Chemicals (Ann Arbor, MI, USA). These chemicals were supplied in acetone, ethanol, or powder form. Chemicals in powder form were directly dissolved in dimethyl sulfoxide (DMSO) and stock concentrations (100 μg/µL or 105 ppm) were prepared. In case of chemicals in acetone or ethanol, they were dried with nitrogen gas under a fume hood and then prepared in stock concentrations (100 μg/µL or 105 ppm) with DMSO. Finally, a working solution (1,000 ppm) was prepared by a serial dilution using DMSO. Fura-8AM was purchased from AAT Bioquest (Sunnyvale, CA, USA) and dissolved in DMSO. Phosphate-buffered saline (PBS) was prepared with 100 mM phosphoric acid and adjusted to pH 7.4.
5
Microfluidic Competitive Chemotaxis Assay
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A Microfluidic Competitive Chemotaxis-Chip (μC3) device (Figure S1
6
Neutrophil Actin Polymerization Assay
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Neutrophils were purified from bone marrow as described previously.10 Briefly, bone marrow‐derived neutrophils were harvested using 62% Percoll. For the actin polymerization assay, purified neutrophils (4 × 105 cells) were suspended in HBSS (Nacalai Tesque) containing 0.2% bovine serum albumin (Sigma Aldrich) and allowed to adhere to fibronectin‐coated coverslips (Neuvitro) for 15 minutes at 37°C in a 5% CO2 incubator. Neutrophils were treated with either 1000, 100, 10, or 1 nmol/L of commercially available Cayman (±)17,18‐EpETE, BM‐3 17(S),18(R)‐EpETE, 18‐HEPE (Cayman Chemical), RvE1 (Cayman Chemical), or 0.03% (vol/vol) ethanol (vehicle control) for 15 minutes and then stimulated with 1 μmol/L N‐formyl‐methionyl‐phenylalanine (fMLP; Sigma Aldrich) or 100 nmol/L leukotriene B4 (LTB4; Cayman Chemical) for 2 minutes at 37°C in a 5% CO2 incubator. Neutrophils were fixed in 4% paraformaldehyde (Nacalai Tesque), permeabilized using 0.5% (vol/vol) Triton X‐100 (Nacalai Tesque) in PBS, and stained with 100 nmol/L Acti‐stain 488–phalloidin (Cytoskeleton) for 30 minutes at room temperature. Finally, cell nuclei were stained by incubating neutrophils with 4ʹ,6‐diamidino‐2‐phenylindole for 30 seconds at room temperature. Images were obtained with Leica TCS SP8 confocal microscopy (Leica Microsystems).
7
Siderophore-Mediated Bacterial Interactions
8
Neutrophil LL-37 Release Assay with SBDS
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The LL-37 assay was adapted from the protocol of Wan et al26 (link) 1×106 Neutrophils (120 µL) in RPMI1640 medium supplemented with 10% FCS were seeded in 96 well plates. The neutrophils (isolated from blood) were preincubated with 0, 10, 50 µg/mL of SBDS for 30 min at 37 °C/5% CO2 atmosphere. Leukotriene B4 (LTB4) of 1 µM (Cayman Chemical, Michigan, USA) was added or the cells were left untreated and incubated for 5 min. The supernatant was collected and LL-37 was determined by ELISA, as recommended by the supplier (Hycultec GmbH, Beutelsbach, Germany). For analysis, the absorbance values from standard and from samples were corrected with that of the samples without cells. The concentration of LL-37 was extrapolated using the standard curve.
9
Immune Cell Isolation and Activation Protocol
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RPMI-1640, Ammonium-Chloride-Potassium (ACK) lysing buffer, EDTA 0.5M, and HEPES 1M were purchased from Lonza (Walkersville, MD). MEM nonessential amino acids (NEAA), L-glutamine, Penicillin/streptomycin, Hanks’ Balanced Salt Solution with Ca2+ and Mg2+ (HBSS+) or HBSS without Ca2+ and Mg2+ (HBSS−), and Phosphate Buffered Saline with (PBS+) or without Ca2+ and Mg2+ (PBS−) were purchased from Corning Cellgro, Mediatech Inc. (Tewksbury, MA). Fetal bovine serum (FBS) was from Atlanta Biologicals (Oakwood, GA). Leukotriene B4 (LTB4) was from Cayman Chemical (Ann Arbor, MI). N-Formylmethionyl-leucyl-phenylalanine (fMLF), DNase I, Histopaques-1077 and −1119, 2,2'-azino-di-(3-ethyl)di-thiazoline-sulfonic-acid (ABTS), DuoLink® In situ-Fluorescence kit, and proteinase inhibitor cocktail (cat#P8340) were from Millipore-Sigma (St. Louis, MO). Recombinant Protein G-Sepharose® 4B, Hoechst 33342, CountBright counting beads, and Prolong antifade mounting agent were from Invitrogen (Carlsbad, CA). Recombinant murine E-selectin-Fc, VCAM-1-Fc and ICAM-1-Fc chimera molecules, and the polyclonal goat anti-mouse CD47 (cat#AF1866) and goat anti-human JAM-A (cat#AF1077) antibodies were from R&D Systems (Minneapolis, MN). Recombinant mouse TNF-α and IFN-γ were purchased from Peprotech (Rocky Hill, NJ). A complete list of antibodies used in this work is provided as supplemental material .
10
Chemokine Gradient Formation Assay
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A blunt needle connected to the inlet of the device served as a loading reservoir for chemokines diluted in migration buffer and the cells. The device was primed 15 minutes prior to cell loading with fMLF (Sigma-Aldrich), leukotriene B4 (LTB4, Cayman Chemicals, Ann Arbor, MI), IL-8 and C5a (R&D Systems Inc, Minneapolis, MN) and/or its combinations with fibronectin (120 kDa, Sigma Aldrich, 1 μg/mL, 1:20 dilution) or with collagen (Corning® Collagen I, Corning Incorporated, Corning, NY). After 15 min, the chemokine solution was gently flushed out of the main channel, resulting in the formation of a chemokine gradient inside the maze.
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