Ultrapure LPS from E. coli 0111:B4, a TLR4 agonist, and the synthetic bacterial lipoprotein (Pam3CSK4), a TLR2/TLR1 agonist, were purchased from Invivogen (San Diego, USA). BD OptEIA human IL-1β, IL-6, MCP-1, TNF-α and IL-10 assay kits were purchased from BD Biosciences (Mississauga, Canada). The FLICA polycaspases kit for measuring apoptosis was purchased from ImmunoChemistry Technologies, LLC (Bloomington, USA). The Griess Reagent System was purchased from Promega (Madison, USA). The human myeloid THP-1 monocyte/macrophage human cell line was maintained in continuous culture with RPMI 1640 with 10 mM l -glutamine (Gibco) supplemented with 10% fetal bovine serum (FBS) US source (Gibco), 100 units per mL penicillin and 100 µg mL–1 streptomycin Pen Strep (Gibco) in an atmosphere of 5% CO2 at 37 °C, as a suspension of cells.
Ultrapure lps from e coli 0111 b4
Manufactured by InvivoGen
Sourced in United States, France
Ultrapure LPS from E. coli 0111:B4 is a laboratory product that provides a purified source of lipopolysaccharide (LPS) derived from the Escherichia coli 0111:B4 strain. LPS is a key component of the outer membrane of Gram-negative bacteria and can be used for various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Farnesol, by Merck Group (2 mentions)
Geranylgeraniol, by Merck Group (2 mentions)
Luria bertani medium, by Merck Group (2 mentions)
Brain heart infusion medium, by Merck Group (2 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin pen strep, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Pam3csk4, by InvivoGen (1 mentions)
Griess reagent system, by Promega (1 mentions)
Ferric chloride hexahydrate, by Merck Group (1 mentions)
Ferrous chloride tetrahydrate, by Merck Group (1 mentions)
Nigericin, by InvivoGen (1 mentions)
Cetyltrimethylammonium bromide ctab, by Merck Group (1 mentions)
Recombinant mouse gm csf, by R&D Systems (1 mentions)
Cytokine elisa, by BD (1 mentions)
Glycerol p phenylenediamine, by Merck Group (1 mentions)
Citric acid, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Recombinant human cytokines, by R&D Systems (1 mentions)
10 protocols using ultrapure lps from e coli 0111 b4
1
TLR Agonist-Induced Immune Response
2
Synthesis and Characterization of Nanomaterials
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Ferrous chloride tetrahydrate (FeCl2·4H2O), ferric chloride hexahydrate (FeCl3·6H2O), aqueous ammonia (NH3(aq)), citric acid, 3-mercaptopropionic acid (3 MPA), tellurium at 200 mesh (Te), sodium (Na), naphthalene (C10H8), and cetyltrimethylammonium bromide (CTAB) were employed as supplied from Sigma/Aldrich, SP, Brazil (new bottles, kept closed in desiccators, analytical grade). Cadmium acetate (Cd(OAc)), sodium hydroxide (NaOH), tetrahydrofuran (THF), and acetone (C3H6O) were employed as supplied from Labsynth, SP, Brazil. All cell culture reagents (cell media, supplements, Trypsin-EDTA) were acquired from ThermoFisher Scientific, SP, Brazil. All plasticware for cell culture was acquired from Corning, SP, Brazil. Mouse recombinant GM-CSF was acquired from R&D Systems, SP, Brazil, while Ultrapure LPS (from E. coli 0111:B4) and Nigericin were acquired from Invivogen, SP, Brazil. 4′, 6-diamidino-2-phenylindole (DAPI), glycerol-p-phenylenediamine, and all remaining salts and solutions were acquired from Sigma-Aldrich, SP, Brazil. APC-conjugated anti-mouse CD11c and fluorescein-5-isothiocyanate (FITC)-conjugated anti-mouse MHC Class II I-Ab antibodies were acquired from ThermoFisher Scientific, SP, Brazil. Cytokine ELISA were from BD Biosciences, SP, Brazil.
3
TLR4 Agonist and Antagonist Modulation of ADMSC Phenotypes
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The TLR4 agonist which was Ultrapure LPS from E. coli 0111:B4 (Cat no: tlrl‐3pelps), and the TLR4 antagonist which was Ultrapure lipopolysaccharide from R. Spheroids (Cat no: tlrl‐prslps), were both purchased from InvivoGen (San Diego, California) and prepared according to the manufacturer's instructions. Pro‐inflammatory and anti‐inflammatory characters were determined according to TLR4 agonist and TLR4 antagonist responses to cytokines. We defined ADMSCs stimulated with 0.5 μg/mL TLR4 agonist as proinflammatory phenotype, ADMSCs treated with 25 μg/mL TLR4 antagonist as anti‐inflammatory phenotype, and naive and untreated ADMSCs as ADMSCs only. Naïve ADMSCs were selected as the control groups.
4
Immunoblotting of Signaling Pathways
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Antibodies raised against STK isoforms and β-actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies against Syk, as well as all phospho antibodies, were from Cell Signaling (Beverly, MA, USA). Ficoll-Paque Plus was from GE Biosciences (Baie-d'Urfé, QC, Canada); endotoxin-free (<2 pg/ml) RPMI 1640 was from Wisent (St-Bruno, QC, Canada). Recombinant human cytokines were from R&D Systems (Minneapolis, MN, USA), and UltraPure LPS (from E. coli 0111:B4) was from InvivoGen (San Diego, CA, USA). Dimethyl sulfoxide (DMSO), N-formyl-methionyl-phenylalanine (fMLP), and phenylmethanesulphonyl fluoride (PMSF) were from Sigma-Aldrich (St. Louis, MO, USA). Diisopropyl fluorophosphate (DFP) was from Bioshop Inc. (Burlington, ON, Canada). The protease inhibitors, aprotinin, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), leupeptin, and pepstatin A, were all from Roche (Laval, QC, Canada). Kinase inhibitors were all purchased through Cedarlane Labs (Mississauga, Canada). All other reagents were of the highest available grade, and all buffers and solutions were prepared using pyrogen-free clinical grade water.
5
Bacterial culture and inflammatory modulation
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Cultures of E. coli (isolate MS499) (16 ) or T. pyogenes (isolate MS249) (17 ), previously collected from the uteri of postpartum cows with persistent uterine disease, were grown overnight in Luria-Bertani medium (Sigma-Aldrich) and brain-heart infusion medium (Sigma-Aldrich) supplemented with 5% FBS, respectively, as described previously (6 (link), 18 (link)). The concentration of bacteria was measured by colony count and suspended to 1 × 108 CFU/ml in sterile PBS (Life Technologies, Paisley, U.K.), followed by centrifugation at 6000 × g for 10 min at 4°C. After washing, bacteria were diluted to 1 × 103 CFU/ml (E. coli) or 1 × 108 CFU/ml (T. pyogenes) in complete medium. Ultrapure LPS from E. coli 0111:B4 was obtained from InvivoGen (Toulouse, France) and used at 100 ng/ml, because this concentration has previously been shown to be optimal for stimulating IL-6 and CXCL8 responses in endometrial cells (14 (link)). Full details of the small molecules used as a part of the inflammatory modulator screening are provided in Supplemental Table I . The isoprenoid alcohols farnesol and geranylgeraniol were obtained from Sigma-Aldrich.
6
Monocyte Isolation and Activation Protocols
7
Melatonin and ATP Modulate Inflammatory Response
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Melatonin and adenosine 5′-triphosphate (ATP) disodium hydrate were obtained from Sigma-Aldrich (St. Louis, USA) and Ultra-Pure LPS (from E. coli 0111: B4) was purchased from InvivoGen (San Diego, USA). Sirtinol was purchased from Tocris (Bristol, UK). Fetal bovine serum (FBS), RPMI 1640 cell culture media, L-Glutamine, penicillin/streptomycin, phosphate buffered saline (PBS), trypsin/EDTA were purchased from Biochrom (Berlin, Germany). All the antibodies used in experiments are given in Supplementary Table 1 .
8
Bacterial Culture and Inflammatory Modulator Screening
9
Nanopipette Delivery of LPS and Aβ
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LPS and sonicated Aβ fibrils were delivered to the cell surface using nanopipette. 1 μg/ml LPS (Ultrapure LPS from E. coli, 0111:B4, Invivogen) and 4 µM total monomer of sonicated Aβ fibrils were loaded into the nanopipette. The nanopipette was controlled by a 3D manipulator (Scientifica microstar) to move down to the target cell until the nanopipette tip slightly touched the cell membrane. Then the nanopipette was withdrawn back by 3 µm away from the cell surface. At this position, a pressure pulse (3 kPa for 5second) was used to deliver both the LPS and the sonicated Aβ fibrils to the cell surface (SFigure 2 ).
10
Immune Cell PAMP Challenge Assay
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Ninety per cent confluent endometrial epithelial cells, stromal cells, or monocyte-derived DCs were challenged with the following PAMPs: dsDNA CpG dsDNA (ODN 2007), ssRNA (ssPolyU Naked), dsRNA Poly(I:C) low-molecular weight (LMW), dsRNA Poly(I:C) high-molecular weight (HMW), with Lipid A or ultrapure LPS from E. coli 0111:B4 as a positive control (all InvivoGen, Toulouse, France) for the times and concentrations indicated in Section “Results .” Prior to challenge, PAMPs were diluted in OPTI-MEM media (Gibco, UK), containing DOTAP (N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate) liposomal transfection reagent (Sigma-Aldrich Ltd.), and incubated for 5 min at room temperature. The DOTAP/PAMP solution was then added to 1 ml of complete media in a well of a 24-well plate, so that the final concentration of DOTAP was 10 µg/ml. Each experiment was performed using cells isolated from at least three independent animals. Supernatants were collected and stored at −20°C, while cells were washed, and cell lysates collected using PhosphoSafe™ Extraction Reagent (EMD Millipore, UK) and stored at −80°C until further processing for immunoblotting.
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