Lipopolysaccharides lps from escherichia coli 0111 b4

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Lipopolysaccharides (LPS) from Escherichia coli 0111:B4 are complex molecules composed of a lipid and a polysaccharide. They are a major component of the outer membrane of Gram-negative bacteria. LPS from E. coli 0111:B4 can be used in various research applications.

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13 protocols using lipopolysaccharides lps from escherichia coli 0111 b4

Stimulation of Chicken Toll-like Receptors

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Pam3CSK4 (synthetic triacylated lipoprotein), polyI:C and R848 were purchased from Invivogen (San Diego, California, USA). Synthetic class A CpG ODN 2216 [5′- GGGGGACGA:TCGTCGGGGGG-3′], synthetic class B CpG ODN 2007 [5′- TCGTCGTTGTCGTTTTGTCGTT- 3′], synthetic class B CpG ODN 1826 [ 5′- TCCATGACGTTCCTGACGTT- 3′], synthetic class C CpG ODN 2395 [5′- TCGTCGTTT-TCG-GCGCGCGCCG- 3′], non-CpG ODN [5′ -TGCTGCTTGTGCTTTTGTGCTT- 3′], lipopolysaccharides (LPS) from Escherichia coli 0111:B4 and LPS from E. coli 026:B6 were purchased from Sigma–Aldrich (Oakville, Ontario, Canada). These ligands were selected as they have previously been shown to stimulate chicken TLRs [18] (link), [19] .

Barjesteh N., Behboudi S., Brisbin J.T., Villanueva A.I., Nagy É, & Sharif S. (2014). TLR Ligands Induce Antiviral Responses in Chicken Macrophages. PLoS ONE, 9(8), e105713.

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Inhibition of PDE4 by Compound A

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Lipopolysaccharides (LPS) from Escherichia coli 0111:B4 was obtained from Sigma (Product number L4391; the same batch 036M4070V was used for both studies). The test-compound A was synthesized at Grunenthal, Aachen, Germany, and the purity of the batch used in this study was ≥ 95%. The physico-chemical properties of test compound A are presented in Table 1. Test-compound A was developed as an inhibitor of PDE4. The rat TNF_α Quantikine ELISA kit was purchased from R&D systems (SRTA00, Batches P143557, P118837, and 339837). All other reagents and chemicals were of analytical grade and were obtained from standard vendors.Table 1

Physico-chemical properties of compound A

Parameter	Value
Molecular weight	< 500 g·mol⁻¹
cLogP	< 2.5
PSA	< 80 Å²
Solubility	> 10 µmol·L⁻¹ at pH 7.4

Held F., Hoppe E., Cvijovic M., Jirstrand M, & Gabrielsson J. (2019). Challenge model of TNFα turnover at varying LPS and drug provocations. Journal of Pharmacokinetics and Pharmacodynamics, 46(3), 223-240.

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LPS-Induced Inflammatory Response in Mice

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6–8 weeks old WT and Tmem178^−/− C57BL/6 male mice (WT n = 12; Tmem178−/− n = 17) were injected with 25 mg/kg Lipopolysaccharides (LPS) from Escherichia coli 0111:B4 (Sigma). Blood was collected by submandibular bleed 2 h after LPS administration, and serum was harvested by centrifugation at 8000g for 5 min at 4° to analyze TNFa levels by ELISA (BD Biosciences).

Yang Z., Yan H., Dai W., Jing J., Yang Y., Mahajan S., Zhou Y., Li W., Macaubas C., Mellins E.D., Shih C.C., Fitzpatrick J.A, & Faccio R. (2019). Tmem178 negatively regulates store-operated calcium entry in myeloid cells via association with STIM1. Journal of autoimmunity, 101, 94-108.

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Melatonin Derivatives Inhibit Inflammation

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Melatonin was purchased from Shanghai Chemical Co. Ltd. (Shanghai, China). The melatonin derivatives, BBM and EBM, were synthesized and characterized by the Melatonin Research Group, Khon Kaen University, Thailand [20 (link)]. RPMI 1640, fetal bovine serum (FBS), antibiotic-antimycotic (100X), 5% trypsin-EDTA, Dulbecco’s phosphate-buffered saline (DPBS) 10X, and 0.4% trypan blue stain were purchased from Gibco. Inc. (New York, NY, USA). Further, 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) was purchased from Invitrogen (Eugene, Oregon, USA). Lipopolysaccharides (LPS) from Escherichia coli 0111:B4, sulfanilamide and naphthylethylenediamine dihydrochloride (NED) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO), and phosphoric acid and acetone were obtained from Fisher Chemical (Loughborough, UK). Mouse PGE₂, IL-6, TNF-α ELISA kits were purchased from R&D Systems, A Bio-Techne (Minneapolis, MN, USA). Aspirin and croton oil were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Sangchart P., Panyatip P., Damrongrungruang T., Priprem A., Mahakunakorn P, & Puthongking P. (2021). Anti-Inflammatory Comparison of Melatonin and Its Bromobenzoylamide Derivatives in Lipopolysaccharide (LPS)-Induced RAW 264.7 Cells and Croton Oil-Induced Mice Ear Edema. Molecules, 26(14), 4285.

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Immune Cell Isolation and Stimulation

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RPMI 1640 medium, fetal bovine serum (FBS), L-glutamine, and penicillin/streptomycin were from Gibco by Life Technologies, UK. Ficoll-Paque Plus was from GE Healthcare, Sweden. Phosphate-buffered saline (PBS) was from HyClone, Logan, Utah. Dimethyl sulfoxide (DMSO) was from Santa Cruz Biotechnology, USA. PP2 (4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d] pyrimidine) was from Sigma Aldrich, USA. Lipopolysaccharides (LPS) from Escherichia coli 0111:B4 were from Sigma Aldrich, USA. BD vacutainer heparinized tubes were from BD Biosciences, UK. Enhanced chemiluminescence (ECL) was obtained from Bio-Rad, USA.

Irtegun S., Pektanc G., Akkurt Z.M., Bozkurt M., Turkcu F.M, & Kalkanli-Tas S. (2016). Pharmacological Inactivation of Src Family Kinases Inhibits LPS-Induced TNF-α Production in PBMC of Patients with Behçet's Disease. Mediators of Inflammation, 2016, 5414369.

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Macrophage Activation by LPS and IFN-γ

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Lipopolysaccharides (LPS) from Escherichia coli 0111:B4 (product no. L3012) were purchased from Sigma-Aldrich; Merck KGaA. Recombinant mouse interferon-γ (IFN-γ; cat. no. 485-MI-100; R&D Systems, Inc.) was used for macrophage intervention. The levels of plasma blood urea nitrogen (BUN; cat. no. C013-2-1) and serum creatinine (SCr; cat. no. C011-2-1) were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's instructions.

Zhang Z., Chen H., Zhou L., Li C., Lu G, & Wang L. (2022). Macrophage-derived exosomal miRNA-155 promotes tubular injury in ischemia-induced acute kidney injury. International Journal of Molecular Medicine, 50(3), 116.

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Synthesis and Characterization of Amino Acid Conjugates

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L-Glutamic acid dimethyl ester hydrochloride, tCA, 1,1′-carbonyldiimidazole (CDI), DNBS, nicotinic acid (NA), trimethylamine (TEA), and SSZ were purchased from Tokyo Chemical Industry (Tokyo, Japan). L-Aspartic acid dimethyl ester hydrochloride, lipopolysaccharides (LPS from Escherichia coli 0111:B4), and mepenzolate (MPZ) were purchased from Sigma Chemical Co., Inc. (St. Louis, MO, USA). The solvents used for synthesis such as ethyl acetate (EA) and acetonitrile (ACN) were purchased from Junsei Chemical Co. (Tokyo, Japan) and DAEJUNG Chemicals & Metals Co., Ltd. (Gyeonggi-do, Republic of Korea). Cytokine-induced neutrophil chemoattractant-3 (CINC-3) and interleukine-10 (IL-10) were quantified using enzyme-linked immunosorbent assay (ELISA) kits obtained from R&D Systems (Minneapolis, MN, USA). The chemicals and solvents were used without additional purification. For the identification of structures, conjugates synthesized were subjected to infrared (IR), proton nuclear magnetic resonance (¹H-NMR), and mass analysis. A Varian FT-IR spectrophotometer (Varian, Palo Alto, CA, USA), a Varian AS 500 NMR spectrophotometer (Varian), and a mass spectrometer (LCMS-8050, Shimadzu Co., Kyoto, Japan) were used for the structural analysis. Tetramethylsilane was used as an internal reference compound to present the chemical shifts in the NMR spectra.

Kang C., Kim J., Ju S., Cho H., Kim H.Y., Yoon I.S., Yoo J.W, & Jung Y. (2022). Colon-Targeted Trans-Cinnamic Acid Ameliorates Rat Colitis by Activating GPR109A. Pharmaceutics, 15(1), 41.

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Evaluation of Inflammatory Responses

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All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, Missouri, United States, Life Technologies (Grand Island, New York, United States), Thermo Scientific (Rockford, Illinois, United States) and Lonza (Walkersville, Maryland, United States). Polymyxin B sulfate was purchased from Santa Cruz Biotechnology (Dallas, Texas, United States), recombinant low endotoxin HSP70 (Cat# ADI-ESP-555-F, < 50 EU/mg purified protein) was purchased from Enzo Life Sciences (Farmingdale, New York, United States). RAW-BlueTM cells (RAW264.7 macrophages stably transfected with the NF-κB reporter gene) were purchased from InvivoGen (San Diego, California, United States). Lipopolysaccharides (LPS) from Escherichia coli 0111:B4 were purchased from Sigma-Aldrich (St. Louis, Missouri, United States).

Dvoriantchikova G., Santos A.R., Saeed A.M., Dvoriantchikova X, & Ivanov D. (2014). Putative role of protein kinase C in neurotoxic inflammation mediated by extracellular heat shock protein 70 after ischemia-reperfusion. Journal of Neuroinflammation, 11, 81.

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Yucamiel Propolis Anti-inflammatory Assays

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Yucamiel propolis paste was used for all experiments. HPLC-MS grade methanol (MeOH), acetonitrile (ACN), and water (H 2 O) from J. T. Baker and reagent grade formic acid (95%) were employed to acidify the mobile phase for chromatography experiments. Ethanol (95%), dexamethasone, trypan-blue dye, dimethyl sulfoxide (DMSO), lipopolysaccharides (LPS) from Escherichia coli (0111: B4), MTT, carrageenan, 12-O-tetradecanoylphorbol 13-acetate (TPA), acetone, and indomethacin were purchased from Sigma Aldrich ® (MO, USA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and penicillin-streptomycin were purchased from Invitrogen Gibco-BRL ® (NY, USA). Murine IL-1β ELISA development kit (900-K47), murine IL-4 ELISA development kit (900-K49), murine IL-6 ELISA development kit (900-K50), murine IL-10 ELISA development kit (900-K53), and murine TNF-α ELISA development kit (900-K54) were obtained from Peprotech ® (London, UK).

Xool-Tamayo J., Chan‐Zapata I., Arana‐Argáez V., Torre F.V.l., Torres‐Romero J.C., Araujo-León J.A., Aguilar-Ayala F.J., Rejón-Peraza M.E., Castro-Linares N., Vargas-Coronado R.F., & Cauich‐Rodríguez J.V. (2020). In vitro and in vivo anti-inflammatory properties of Mayan propolis. European Journal of Inflammation, 18, 205873922093528-205873922093528.

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Comprehensive Reagents Acquisition Protocol

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Chemicals. All chemicals were purchased from Sigma-Aldrich or Tokyo Chemical Industry (TCI) unless otherwise stated. Cisplatin, gentamicin, diatrizoate, alpha-naphthyl isothiocyanate (ANIT) and lipopolysaccharides (LPS, from Escherichia coli 0111:B4) were obtained from Sigma-Aldrich. N-acetyl-β-D-glucosaminidase, plasmin, β-galactosidase, nitroreductase and fibroblast activation protein-alpha were purchased from Sigma-Aldrich.
Alanine aminopeptidase, furin and recombinant human caspase-3 were purchased from R&D Systems. Creatinine assay kit, urea assay kit and mouse cystatin C ELISA kit were purchased from Sigma-Aldrich, BioAssay Systems and RayBiotech, respectively. Mouse NGAL, osteopontin, KIM-1, TFF3 and clusterin ELISA kit were purchased from R&D Systems. Cleaved-caspase-3 antibody (9661L) was purchased from Cell Signaling Technology. Mouse beta2-microglobulin ELISA kit, anti-CD31 antibody (ab28364), anti-CD11b antibody (ab133357) and Alexa Fluor 488 conjugated goat anti-rabbit IgG H&L (ab150077) were purchased from Abcam. Ultrapure water was supplied by Milli-Q Plus System (Millipore Corporation, Breford, USA).

Huang J., Li J., Lyu Y., Miao Q, & Pu K. (2019). Molecular optical imaging probes for early diagnosis of drug-induced acute kidney injury. Nature materials, 18(10).

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