Reagents and instruments used in this study were High Performance Liquid Chromatography combined with Diode Array Detector (Agilent 1100 Series, USA); AG285 electronic analytical balance (Mettler-Toledo, Switzerland); Microplate Reader (Bio-Tek FLx800, USA); XDA-1 macroporous resin (LanxiaoTechnology Companies, Xi’an, China); Complete Freund’s Adjuvant (Sigma, USA; Lot SLBN5308V; PCode: 1002093124); indomethacin and methotrexate (China Pharmaceutical Co., Ltd., Shanghai); tripterygium glycosides (TGs, Xiahua Pharmaceutical Industry, Shanghai, China; Lot No.: 160903); Assay kits for IL-6, IL-1β, IL-17 and TNF-α (Ebioscience, USA). Fetal bovine serum (FBS), α-Modified minimal essential medium (α-MEM), phosphate buffered saline (PBS) and penicillin/streptomycin were obtained from Gibco company (USA). Dimethyl sulfoxide (DMSO) was purchased from WAK-Chemie Medical GmbH (Steinbach, Hesse, Germany). Antibodies against IKBα, JUNK, P-ERK, ERK and p-38 were purchased from Boster Biological Technology (Wuhan, China). All the other antibodies used in this study were obtained from Cell Signaling Technology (Beverly, MA, USA). Lipopolysaccharide (LPS) from Escherichia coli 055:B5 was purchased from Sigma-Aldrich Company (USA). The BCA kit for assay of protein was purchased from Biyotime (Shanghai, China).
Lipopolysaccharide lps from escherichia coli 055 b5
Manufactured by Merck Group
Sourced in United States
Lipopolysaccharide (LPS) from Escherichia coli 055:B5 is a bacterial endotoxin that is used as a research tool. It is derived from the cell wall of the Gram-negative bacterium Escherichia coli 055:B5.
Automatically generated - may contain errors
Lab products found in correlation
Diode array detector, by Agilent Technologies (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
P erk, by Boster Bio (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Complete freund s adjuvant, by Merck Group (1 mentions)
Polyinosinic polycytidylic acid poly 1 c, by InvivoGen (1 mentions)
Sulfanilamide, by Thermo Fisher Scientific (1 mentions)
Petroleum ether, by Merck Group (1 mentions)
Monosaccharide standard, by Merck Group (1 mentions)
E toxate kit, by Merck Group (1 mentions)
Dulbecco s modified eagle medium, by Merck Group (1 mentions)
Mouse il 6 elisa kit, by Abcam (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by GE Healthcare (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Penicillin streptomycin, by GE Healthcare (1 mentions)
Amphotericin b, by GE Healthcare (1 mentions)
D glucose, by Merck Group (1 mentions)
Mouse tnf α, by R&D Systems (1 mentions)
Penicillin streptomycin amphotericin, by Thermo Fisher Scientific (1 mentions)
12 protocols using lipopolysaccharide lps from escherichia coli 055 b5
1
Biological Evaluation of Tripterygium Glycosides
2
Synthetic Peptides and Adjuvants for Immunotherapy
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Synthetic peptides were purchased from Genecust (Dudelange, Luxembourg) and dissolved in 10% dimethyl sulfoxide in phosphate-buffered saline (PBS). The purity of the peptides was >80%. Montanide was provided by SEPPIC and Berna-Biotech (Bern). Polyinosinic‐polycytidylic acid (Poly IC) was purchased from InvivoGen (San Diego, California, USA) and was diluted in PBS before injection. Poly-ICLC (Hiltonol, a synthetic poly-IC, stabilized with polylysine and carboxymethylcellulose) was kindly provided by Dr Andrés Salazar (Oncovir, NW Washington, District of Columbia, USA). Lipopolysaccharide (LPS) from Escherichia coli 055:B5 was purchased from Sigma (Madrid, Spain).
The hEDA-HPVE7-16 and hEDA-HPVE7-16/18 were produced in E. coli BL21 using a T7 expression vector and purified from inclusion bodies by size exclusion chromatography (Biotecnol, Oeiras, Portugal). The mEDA-HPVE7-16 protein was produced at CIMA as previously described.15 (link) The levels of endotoxin were below 0.1 EU/μg protein as tested by Quantitative Chromogenic Limulus Amebocyte Lysate assay (Cambrex, Walkersville, Maryland, USA).
The hEDA-HPVE7-16 and hEDA-HPVE7-16/18 were produced in E. coli BL21 using a T7 expression vector and purified from inclusion bodies by size exclusion chromatography (Biotecnol, Oeiras, Portugal). The mEDA-HPVE7-16 protein was produced at CIMA as previously described.15 (link) The levels of endotoxin were below 0.1 EU/μg protein as tested by Quantitative Chromogenic Limulus Amebocyte Lysate assay (Cambrex, Walkersville, Maryland, USA).
3
Macrophage Phagocytosis Assay Protocol
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N-1-napthylethylenediamine dihydrochloride and sulfanilamide were purchased from Acros Organics (Geel, Belgium). Dimethyl sulfoxide, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide], Dulbecco's modified Eagle medium, E-TOXATE™ kit, monosaccharide standards (arabinose, xylose, fucose, rhamnose, galactose and mannose) and lipopolysaccharide (LPS) from Escherichia coli 055:B5 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Ethanol, petroleum ether, and D-(+)-glucose were purchased from Merck (Darmstadt, Germany). Fetal bovine serum, penicillin/streptomycin, and amphotericin B were from PAA Laboratories (Cölbe, Germany). The Phagocytosis Assay Kit IgG fluorescein isothiocyanate (FITC) and the iNOS polyclonal antibody were purchased from Cayman Chemical (Ann Arbor, MI, USA). The TNF-α mouse ELISA kit and the IL-6 mouse ELISA kit were obtained from Abcam (Cambridge, UK). Diethylaminoethyl cellulose pre-swollen ion exchanger was purchased from Whatman (Maidstone, England). A 2-D protein extraction buffer was purchased from GE Healthcare (Piscataway, NJ, USA). β-actin polyclonal antibodies were purchased from BioVision Research Instrument (Milpitas, CA, USA). Donkey anti-rabbit IgG was obtained from Abnova (Taipei City, Taiwan).
4
Alisol F and 25-anhydroalisol F Isolation and Effects
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Alisol F and 25-anhydroalisol F were isolated from A. orientale by one of the authors (Q. Ma) using our previously-established method and provided with a purity of 98.0% determined by high-pressure liquid chromatography. Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), 0.25% trypsin, and penicillin-streptomycin-amphotericin (PSA) were purchased from Gibco BRL Co. Ltd. (Gaithersbug, MD, USA). Lipopolysaccharide (LPS) from Escherichia coli 055:B5, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), dexamethasone (DXM), d -galactosamine (d -gal) and Trizol reagent were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The Griess reagent, the protein extraction kit and BCA protein assay kit were obtained from Beyotime Institute of Biotechnology (Beijing, China). Go Tag® qPCR Master Mix and GoScriptTM Reverse Transcription System were purchased from Promega (Madison, WI, USA). Rabbit polyclonal antibodies against inducible nitric oxide synthase (iNOS), COX-2, p-p38 (Thr180/Tyr182), p38, p-ERK1/2 (Thr202/Try204), ERK1/2, p-SAPK/JNK (Thr183/Tyr185), SAPK/JNK, p-p65 (Ser536), p65, GAPDH, p-IκB-α (Ser32), IκB-α, p-STAT3 (Tyr705), STAT3, goat-IgG HRP, and lamin B1 were purchased from Cell Signaling Technology (Danvers, MA, USA). Mouse TNF-α, IL-1β, and IL-6 ELISA kit were from R and D systems (Abingdon, UK).
5
Inflammatory Cytokine Detection Assay
6
Differentiation of U937 Monocytes to Macrophages
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Linagliptin compounds were provided by Boehringer Ingelheim Pharmaceuticals, Inc. (Ingelheim am Rhein, Rhineland-Palatinate, Germany); ketoprofen and loxoprofen sodium salt dihydrate (Loxo) and interleukin (IL)-1β from Wako Pure Chemical Industries Ltd. (Osaka, Japan); and lipopolysaccharide (LPS) from Escherichia coli 055:B5 from Sigma-Aldrich (St. Louis, MO, USA) and phorbor 12-myristate 13-acetate (PMA) from abcam. All chemicals used in this study were of the purest grade available commercially.
Human U937 monocytes (EC85011440) were purchased from the European Collection of Animal Cell Culture UK. All the following operations were carried out under sterile conditions. The cells at densities of 1 × 106 cells/mL were cultured via routine methods in Roswell Park Memorial Institute medium (RPMI 1640) (Wako Pure Chemical Industries Ltd., Osaka, Japan) containing L-glutamine and phenol red with 10% of fetal bovine serum (FBS) (Biosera, NUAILLE, France), 100 nM PMA, 1% of antibiotic-antimycotic solution from gibco by Life Technologies (Carlsbad, CA) at 37 °C, and 5% CO2 for 48 h for differentiation into macrophages. The cells were then subsequently used in the experiment.
Human U937 monocytes (EC85011440) were purchased from the European Collection of Animal Cell Culture UK. All the following operations were carried out under sterile conditions. The cells at densities of 1 × 106 cells/mL were cultured via routine methods in Roswell Park Memorial Institute medium (RPMI 1640) (Wako Pure Chemical Industries Ltd., Osaka, Japan) containing L-glutamine and phenol red with 10% of fetal bovine serum (FBS) (Biosera, NUAILLE, France), 100 nM PMA, 1% of antibiotic-antimycotic solution from gibco by Life Technologies (Carlsbad, CA) at 37 °C, and 5% CO2 for 48 h for differentiation into macrophages. The cells were then subsequently used in the experiment.
7
Murine Macrophage Cultivation and Treatment
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RAW 264.7 murine macrophage cells were obtained from the American Type Culture Collection (Manassas, VA, USA). The macrophages were cultured in high-glucose Dulbecco's Modified Eagle's medium (DMEM; Welgene Inc., Daegu, South Korea) supplemented with 10% (v/v) fetal bovine serum (FBS; Grand Island, NY, USA) and antibiotics (10 μg/mL streptomycin, and 100 IU/mL penicillin) at 37°C and 5% CO2 in a humidified atmosphere of 95% air. These cells were cultured to ≥85% confluence before treatment with empagliflozin (AdooQ Bioscience, Irvine, CA), gemigliptin (LG Life Sciences Ltd., Seoul, South Korea), lipopolysaccharide (LPS) from Escherichia coli 055:B5 (Sigma-Aldrich, St. Louis, MO, USA), or dexamethasone (Sigma-Aldrich). empagliflozin and gemigliptin were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) and added to the cell culture at the desired concentrations. The final DMSO concentration did not exceed 0.1%, and all samples were incubated with the same amounts of DMSO. LPS and dexamethasone were dissolved in phosphate-buffered saline (PBS; Welgene Inc.), and all additives were used as cotreatments following a 2-h period of cell starvation.
8
Anti-Inflammatory Effects of Rhein
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Rhein were purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The purity of the compound rhein was certified to be 98% by high-performance liquid chromatography (HPLC). Lipopolysaccharide (LPS) from Escherichia coli 055:B5 was obtained from Sigma-Aldrich Chemical Co. (USA). TNF-α, IL-1β, monocyte chemoattractant protein (MCP)-1 and IL-8 ELISA Kits were purchased from Yantai Science & Biotechnology (Shandong, China). Antibodies against phospho-IκBα, phospho-NF-κB p65 and phospho-IKKβ were obtained from Cell Signaling Technology (Beverly, MA, USA). Blood Urea Nitrogen (BUN) and Serum Creatinine Determination (SCr) assay kit reagents were supplied by were purchased from the Institute of Jiancheng Bioengineering (Nanjing, China). DMEM medium and fetal bovine serum (FBS) were products of Gibco Corporation (USA). Zymosan A, nitroblue tetrazolium (NBT) and the other reagents were all purchased from Sigma-Aldrich Chemical Co. (USA). All other reagents were of analytical grade.
9
Molecular Mechanisms of Erythropoietin Signaling
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Recombinant human EPO (rhEPO) was purchased from Sunshine Pharmaceutical (Shenyang, China). Lipopolysaccharide (LPS from Escherichia coli 055:B5) was purchased from Sigma–Aldrich (St. Louis, MO, United States). EPO mimetic peptide 9 (EMP9, an EPOR antagonist) was synthesized by Fenghui Biotechnology (Changsha, China). Fedratinib (a JAK2 inhibitor), NSC 74859 (a STAT3 inhibitor), and BAY 11-7082 (an NF-κB inhibitor) were from MedChemExpress (Deer Park Dr, NJ, United States). The interleukin-1β (IL-1β), interleukin-18 (IL-18), myeloperoxidase (MPO) enzyme-linked immunosorbent assay (ELISA) kits, and anti-IL-1β antibody (catalog no. AF-401-NA) were from R&D Systems (Minneapolis, MN). Antibodies against phospho-NF-κB p65 (Ser536, catalog no. 3033S), NF-κB p65 (catalog no. 8242S), phospho-STAT3 (Tyr705, catalog no. 9145S), STAT3 (catalog no. 9139S), phospho-JAK2 (Tyr1007/1008), and JAK2 were obtained from Cell Signaling Technology (Beverly, MA, United States). Anti-Caspase-1 (catalog no. AG-20B-0042-C100), anti-NLRP3 (catalog no. AG-20B-0006-C100), and anti-ASC antibodies (catalog no. AG-25B-0006-C100) were from AdipoGen (San Diego, CA, United States).
10
Osthole Modulates Inflammatory Response
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Osthole (purity=98%, HPLC) was brought from Shanxi Scidoor Hi-tech Biology Co, Ltd (Batch number 20120220; Xi’An, China). NF-κB p65 ELISA kit (cat. no. NBP2-31042) was gained fromNovus Biologicals. Assay kit reagents of Serum Creatinine Determination (SCr) and Blood Urea Nitrogen (BUN) were brought from the Institute of Jiancheng Bioengineering (Nanjing, China). Sigma-Aldrich Chemical Co. (America) provided Lipopolysaccharide (LPS) from Escherichia coli 055:B5. ELISA Kits of IL-6, TNF-α, IL-1β, IL-8 and MCP-1 were brought from Yantai Science & Biotechnology (Shandong, China). Fetal bovine serum (FBS) and DMEM medium were products of Gibco Corporation (USA). Nitroblue tetrazolium (NBT), Zymosan A and the other reagents were brought from Sigma-Aldrich Chemical Co. (America). All the other reagents belonged to analytical grade were brought from Sigma-Aldrich Chemical Co. (USA).
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