To investigate the effect of VL-01 on LPS induced cytokine response, mice were i.v. treated with 25 mg/kg VL-01 2 h prior to LPS treatment (Lipopolysaccharides from Escherichia coli 055:B5, Sigma, Germany, 20 µg/mice). Serum samples for cytokine analysis were collected before (−4 h) and 1.5 and 3 h after LPS treatment.
Lipopolysaccharides from escherichia coli 055 b5
Manufactured by Merck Group
Sourced in United States
Lipopolysaccharides from Escherichia coli 055:B5 is a laboratory reagent used in scientific research. It is a complex of lipids and polysaccharides derived from the outer membrane of the Escherichia coli 055:B5 strain. The product is supplied in a purified form for use in various applications in the life sciences and biomedical fields.
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Lab products found in correlation
Normal goat serum (ngs), by Agilent Technologies (1 mentions)
Neomycin sulfate, by Merck Group (1 mentions)
Acetic acid, by Thermo Fisher Scientific (1 mentions)
Ketoconazole, by Merck Group (1 mentions)
Sq22536, by Merck Group (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Bovine calf serum (bcs), by Thermo Fisher Scientific (1 mentions)
Bromoenol lactone, by Cayman Chemical (1 mentions)
Forskolin, by Merck Group (1 mentions)
And gapdh, by Cell Signaling Technology (1 mentions)
Heneicosanoic acid, by Nu-Chek Prep (1 mentions)
Methyl arachidonyl fluorophosphonate mafp, by Cayman Chemical (1 mentions)
2 tedc, by Bio-Techne (1 mentions)
Anti iba1 antibody, by Fujifilm (1 mentions)
Indomethacin, by Merck Group (1 mentions)
Acetonitrile, by Avantor (1 mentions)
Hplc grade water, by Avantor (1 mentions)
Human igg, by Jackson ImmunoResearch (1 mentions)
M peg silane, by Creative PEGWorks (1 mentions)
Alexa fluor 488 donkey anti human igg, by Jackson ImmunoResearch (1 mentions)
8 protocols using lipopolysaccharides from escherichia coli 055 b5
1
VL-01 Modulates LPS-Induced Cytokines
2
Microglia and Astrocyte Activation Assay
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Dulbecco’s Modified Eagle Medium/Ham’a F12 (DMEM/F12 1:1) and bovine calf serum were purchased from Life Technologies Corporation (Carlsbad, CA, USA). Ketoconazole, Indomethacin, SQ 22536, H-89, lipopolysaccharides from Escherichia coli 055:B5 and forskolin were purchased from Sigma-Aldrich (St. Louis, MO, USA), and normal goat serum was acquired from Dako (Carpentaria, CA, USA). Anti-Iba-1 antibody and anti-glial fibrillary acidic protein (GFAP) antibody were purchased from Wako (Richmond, VA, USA) and Sigma-Aldrich (St. Louise, MO, USA), respectively. Antibodies to PKA, CREB, and GAPDH were purchased from Cell Signaling. Heneicosanoic acid was from Nu-Chek Prep (Elysian, MN). Butylated hydroxytoluene (BHT), LY311727, neomycin sulfate, and 10% boron trifluoride-methanol (BF3/methanol) were from Sigma-Aldrich (St. Louis, MO). Methyl arachidonyl fluorophosphonate (MAFP), bromoenol lactone (BEL), and ZLJ-6 were obtained from Cayman Chemical (Ann Arbor, MI). 2-TEDC was from Tocris Bioscience (Bristol, UK). HPLC grade water and acetonitrile were from Avantor Performance Materials (Center Valley, PA). Other solvents, BCA protein assay kit, and acetic acid were from Fisher Scientific (Pittsburgh, PA)
3
Lipopolysaccharide and Taurine Protocol
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Lipopolysaccharides from Escherichia coli (055:B5) and taurine (TAU, ≥99%, T0625-500G) were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). All other reagents used in the experiments were of analytical grade and of highest purity.
4
Immune Response Cytokine Quantification
5
Murine Glioma and Macrophage Proliferation Assay
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Murine GL261 glioma cells and the RAW 264.7 macrophage cell line were obtained from the Developmental Therapeutics Program (DTP), Division of Cancer Treatment and Diagnosis (DCTD) Tumor Repository (National Institutes of Health (NIH), Frederick, MD) and American Type Culture Collection (ATCC), Manassas, Virginia, respectively, and cultured in Dulbecco’s modified Eagle’s medium (Cellgro) with 10% FBS (Cellgro) and antibiotics (50 U/ml penicillin and 50 μg/ml streptomycin) in a humidified atmosphere of CO2/air (5%/95%) at 37°C.
For macrophage polarization, RAW 264.7 cells were cultured in complete medium and stimulated for 24 hours with 1 ng/ml lipopolysaccharides from Escherichia coli 055:B5 (Sigma, St. Louis, Missouri) and 10 IU/ml Interferon gamma (IFN-γ) (Peprotech, Rocky Hill, New Jersey) for M1 or 20 ng/ml Interleukin 4 (IL-4) (R&D Systems, Minneapolis, Minnesota) for M2 activation.
GL261 and RAW 264.7 cell lines were treated with AP20187 with different concentrations (0.01, 0.1, 1, and 10 nM) or vehicle for 24 or 48 hours, and cell proliferation was measured using CyQUANT Direct Cell Proliferation Assay Kit (Life Technologies, Grand Island, New York) according the manufacturer’s protocol.
For macrophage polarization, RAW 264.7 cells were cultured in complete medium and stimulated for 24 hours with 1 ng/ml lipopolysaccharides from Escherichia coli 055:B5 (Sigma, St. Louis, Missouri) and 10 IU/ml Interferon gamma (IFN-γ) (Peprotech, Rocky Hill, New Jersey) for M1 or 20 ng/ml Interleukin 4 (IL-4) (R&D Systems, Minneapolis, Minnesota) for M2 activation.
GL261 and RAW 264.7 cell lines were treated with AP20187 with different concentrations (0.01, 0.1, 1, and 10 nM) or vehicle for 24 or 48 hours, and cell proliferation was measured using CyQUANT Direct Cell Proliferation Assay Kit (Life Technologies, Grand Island, New York) according the manufacturer’s protocol.
6
Macrophage Activation and Nitric Oxide
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Cell culture media, RPMI 1640 with (1.15 mM L-Arginine) or without L-Arginine were obtained from GIBCO and Sigma-Aldrich, respectively. Lipopolysaccharides from Escherichia coli 055:B5, recombinant human IFN-γ produced in E.coli, the Griess reagent and Phorbol-12-myristate -13- acetate (PMA) were all purchased from Sigma-Aldrich. Fetal bovine serum (FBS) was purchased from GIBCO.
7
Molecular Mechanisms of Anti-inflammatory Signaling
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Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and other tissue culture reagents were purchased from Gibco BRL Co. (Grand Island, NY, USA). Lipopolysaccharides from Escherichia coli 055:B5 was purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Primary antibodies, including mouse/goat/rabbit anti-COX-2 (sc-1745), anti-iNOS (sc-650), anti-β-actin (sc-47778), anti-IкB-α (sc-371), anti-phospho-IкB-α (sc-8404), anti-p50 (sc-7178), anti-p65 (sc-8008), and anti-proliferating cell nuclear antigen (PCNA) (sc-7907), and secondary antibodies were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). Anti-phospho-ERK (#9101), anti-ERK (#9102), anti-phospho-JNK (#9251), anti-JNK (#9252), anti-phospho-p38 (#9211), and anti-p38 (#9212) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA) [37 (link)].
8
Fluorescent Microbial Particles and Opsonins
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Heat killed E. coli (E2861- Escherichia coli (K-12 strain) BioParticles, fluorescin conjugate (ex 494/em518), heat killed S. aureus (S-2851-Staphylococcus aureus (Wood strain without protein A) BioParticles®, fluorescein conjugate (ex 494/em518), respective opsonizing reagents (E2870 - E. coli, S-2860 - S. aureus), dextran beads (yellow-green fluorescent Fluor-Spheres (F8852 Invitrogen; ex488nm/518 nm) and DAPI were obtained from Invitrogen. Morphine-HCl powder as well as 75 mg slow release pellets were a generous gift from NIDA (National Institute of Drug Abuse). Lipopolysaccharides from Escherichia coli 055:B5 (cat# L2880) and Lipoteichoic acid from Staphylococcus aureus (cat# L2515) were obtained from Sigma Aldrich. GFP E.coli (gift from Dr. Sundaram Ramakrishnan), Streptococcus pneumoniae (S. pneumoniae) serotype 3 was obtained from ATCC (cat# 6303; Rockville, MD). S. pneumoniae was grown overnight at 37 °C by streaking onto a blood agar plate (Becton, Dickinson and Co.). The colonies were picked, inoculated into brain heart infusion (BHI) broth and incubated for 6 h at 37 °C to produce log-phase organisms. The concentration of bacteria was determined spectrophotometrically (OD590) and confirmed by plating onto blood agar plates.
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