LPS (Escherichia coli, O127:B8) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Mas receptor antagonist A779 was obtained from AbBiotech (San Diego, CA, USA). ACE2 inhibitor MLN-4760 was a product from EMD Millipore (Darmstadt, Germany). SB203580 (a specific inhibitor of p38 MAPK), PD98059 (a specific inhibitor of ERK1/2) and SP600125 (a specific inhibitor of JNK) were purchased from Santa Cruz Biotechnology (Delaware, CA, USA). Rabbit anti-ACE2, anti-p50, anti-p65, and mouse anti-phospho-p50, anti-phospho-p65 and anti-IκBα antibodies were procured from Santa Cruz Biotechnology. Rabbit anti-p38 MAPK, anti-phospho-p38 MAPK, anti-ERK1/2, anti-phospho-ERK1/2, anti-/JNK, anti-phospho-JNK, horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG, and horse anti-mouse IgG antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). TNF-α and IL-1β kits were purchased from Invitrogen (Eugene, OR, USA). AngII and Ang-(1-7) enzyme-linked immunosorbent assay (ELISA) kits were from Kamiya Biomedical (Seattle, WA, USA).
Lps escherichia coli o127 b8
Manufactured by Merck Group
Sourced in United States
LPS (Escherichia coli; O127:B8) is a lipopolysaccharide extracted from the cell wall of the Escherichia coli bacterial strain O127:B8. It is a commonly used endotoxin for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Ibuprofen ibu, by Merck Group (3 mentions)
Gypenosides, by Merck Group (2 mentions)
Mln 4760, by Merck Group (1 mentions)
Anti iκbα, by Santa Cruz Biotechnology (1 mentions)
Anti phospho p38 mapk, by Cell Signaling Technology (1 mentions)
Sp600125, by Santa Cruz Biotechnology (1 mentions)
Anti erk1 2, by Cell Signaling Technology (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Horse anti mouse igg, by Cell Signaling Technology (1 mentions)
Sb203580, by Santa Cruz Biotechnology (1 mentions)
Pd98059, by Santa Cruz Biotechnology (1 mentions)
Anti phospho p65, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti p38 mapk, by Cell Signaling Technology (1 mentions)
Il 1β kits, by Thermo Fisher Scientific (1 mentions)
Anti p65, by Santa Cruz Biotechnology (1 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase hrp conjugated goat anti rabbit igg, by Cell Signaling Technology (1 mentions)
Anti phospho jnk, by Cell Signaling Technology (1 mentions)
Anti jnk, by Cell Signaling Technology (1 mentions)
Raw264, by American Type Culture Collection (1 mentions)
16 protocols using lps escherichia coli o127 b8
1
Investigating Inflammatory Signaling Pathways
2
RAW264.7 cells treatment with PL and LPS
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DMEM (Gibco, Germany) with 10% fetal bovine serum (FBS, Sigma, USA) was used to culture RAW264.7 (ATCC, USA) cells at a 37 °C and 5% CO2 incubator. RAW264.7 cells were seeded on six-well plates with 1 × 106 cells/well and incubated at 37 °C with 5% CO2 until 60–70% confluence was reached. The control group was treated with dimethyl sulfoxide (DMSO, 0.25 µL/mL), used for dilution of PL. The PL group was treated with 2.5 µM piperlongumine (PL, 20,069–09-4, MCE, USA). The LPS group was treated with 1 µg/mL lipopolysaccharide (LPS, Escherichia coli O127:B8, Sigma, USA) for 6 h [26 (link)]. The LPS + PL group was treated with 2.5 µM PL for 2 h, followed by 1 µg/mL LPS for 6 h.
3
Intracerebroventricular LPS Administration in Rats
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Gypenosides, ibuprofen (IBU), and LPS (Escherichia coli; O127:B8) were obtained by Sigma-Aldrich Company (Sigma-Aldrich Co., St. Louis, MO, USA). LPS administrated intracerebroventricularly (i.c.v.) into the lateral ventricle of the rat brain according to the method of Guo et al. [33 (link)] controlled anesthesia with 50 mg/kg sodium pentobarbital intraperitoneal injection (i.p.). Injection of LPS was delivered at a rate of 2 μL/1 min (total 5 min) and injection needles were left in place an additional 5 min. The sham control animals were administrated the vehicle (i.c.v. and i.p.) instead of one of the drug solutions.
4
Co-culture Model of Intestinal Epithelium and Macrophages
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Caco-2 cells (passages 10 to 12) were seeded at a density of 4.76 × 105 cells/cm2 in polyethylene terephthalate cell culture inserts with a pore size of 0.4 μm (BD Falcon, VWR, Denmark) and placed in 6-well cell culture plates (BD Falcon, VWR, Denmark). Cells were cultured for 21 days. On day 20, RAW264.7 cells (passages 5 to 8) were seeded in 6-well cell culture plates at a density of 1.04 × 105 cells/cm2 in 10% FCS cell culture media. To establish the co-culture model, Caco-2 inserts were transferred to RAW264.7 wells on day 21. RAW264.7 cells were stimulated with 1 μg/ml LPS (Escherichia coli O127:B8, Sigma-Aldrich) whereas 0.001, 0.1, 10, and 100 ng/ml PKD1 (129.3 kDa, Thermo Fisher Scientific, Denmark) were added apically to Caco-2 cells (Figure 1 ). Two studies were performed, either LPS was administered for 3 h followed by PKD1 for 3 h or PKD1 was administered for 3 h followed by LPS for 6 h. Supernatants were collected and stored at −80°C for ELISA analysis, while cells were harvested and stored in RNAlater at −80°C for qPCR analysis. Three biological replicas were performed.
5
Immunomodulatory Responses of BEAS-2B and THP-1 Cells
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SBDE, LPS (Escherichia coli O127:B8; Sigma), and PGN
(Staphylococcus aureus; Sigma) treatments were performed on
BEAS-2B and THP-1 cell lines. Ligand stimulants (SBDE, LPS, PGN) were prepared
by dissolving stock concentrations in a serum-free culture medium to achieve a
final concentration of 10 µg/ml (LPS and PGN) and 5% (SBDE), respectively. Five
percent SBDE is known to induce maximal pro-inflammatory response with limited cytotoxicity.13 (link) Stimulant treatments were performed at the respective final
concentrations and media alone treated cells served as no treatment controls.
Stimulant and no treatment control cell pellets were prepared at 0 − and 24-h
post treatment for kinome and transcriptional analyses.
(Staphylococcus aureus; Sigma) treatments were performed on
BEAS-2B and THP-1 cell lines. Ligand stimulants (SBDE, LPS, PGN) were prepared
by dissolving stock concentrations in a serum-free culture medium to achieve a
final concentration of 10 µg/ml (LPS and PGN) and 5% (SBDE), respectively. Five
percent SBDE is known to induce maximal pro-inflammatory response with limited cytotoxicity.13 (link) Stimulant treatments were performed at the respective final
concentrations and media alone treated cells served as no treatment controls.
Stimulant and no treatment control cell pellets were prepared at 0 − and 24-h
post treatment for kinome and transcriptional analyses.
6
Telmisartan Modulates LPS-Induced Inflammation
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The bronchial BEAS-2B epithelial cells were purchased from ATCC (Maryland, USA) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 5% fetal bovine serum (FBS) at 37°C and 5% CO2. The treatment reagents LPS (Escherichia coli O127:B8) and Telmisartan (98% purity, HPLC grade) were purchased from Sigma-Aldrich (St. Louis, USA). The concentration of Telmisartan was optimized with a series of dose-response experiments for 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) and LDH experiments (0, 1, 2, 10, 20, 100, and 200 μM) for 24 hours. For all other experiments, cells were challenged with LPS (30 μg/ml) with or without Telmisartan (10, and 20 μM) for 24 hours.
7
Isolation and Culture of Human Monocytes
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PBMCs of anonymous healthy donors were isolated from buffy coats purchased from the Beijing Red Cross Blood Center using density gradient cell separation by Ficoll (Lymphoprep; STEMCELL Technologies) following the protocol approved by the institutional review board of the School of Medicine, Tsinghua University. The private information of anonymous blood donors was inaccessible to investigators. PBMCs of RA patients were obtained from Peking Union Medical College Hospital using the protocol that was approved by the institutional review board of Peking Union Medical College Hospital. CD14+ monocytes were further isolated from PBMCs using anti-CD14 magnetic beads (130-050-201; Miltenyi Biotec). CD14+ monocytes were cultured in RPMI 1640 medium (10040CM; Corning) supplemented with 10% (vol/vol) FBS (Gibco) and human recombinant M-CSF (300-25, 10 ng/ml; PeproTech). LPS (Escherichia coli O127:B8; Sigma-Aldrich), human recombinant IL-7 (200-07; PeproTech), human recombinant TNF (H8916; Sigma-Aldrich), or chemical inhibitors (SB203580 from Selleck; STAT5 inhibitor from Santa Cruz Biotechnology; and Bay 11-7082 from Sigma-Aldrich) were used as indicated for various experiments.
8
Annexin A1-derived Peptide and Piplartine in Cell Culture
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The annexin A1-derived peptide Ac2-26 (Ac-AMVSEFLKQAWFIENEEQEYVQTVK)83 (link) was obtained from THERMO SCIENTIFIC (Waltham, MA, USA) and dissolved in dimethyl sulfoxide (DMSO) at a final concentration in culture medium of 1uM. Piplartine (C17H19NO5; CAS number 20069-09-4) was isolated as previously described36 (link) and dissolved in DMSO at a final concentration in culture medium of 10 or 20 µM. PL was also dissolved in absolute ethyl alcohol for UV–Vis absorbance and fluorescence spectroscopy assays. Ac2-26 and PL concentrations were determined by experiments previously performed by our group20 (link),84 (link) and by other authors85 (link)–87 (link). Bacterial lipopolysaccharide (LPS, Escherichia coli O127:B8, SIGMA ALDRICH, Poole, Dorset, UK), a component of the surface membrane of most gram-negative bacteria and potent stimulator of immune cells, was diluted in 10% MEM-Earle medium (CULTILAB, Campinas, SP, Brazil) at a final concentration in culture medium of 10 µg/mL.
9
Intracerebroventricular LPS Injection
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Carvacrol, ibuprofen (IBU), and LPS (Escherichia coli; O127:B8) were purchased from Sigma-Aldrich Company (Sigma-Aldrich Co., St. Louis, MO, USA). LPS was administered intracerebroventricularly into the lateral ventricle of the rat brain according to the method of Guo et al. [26 (link)] with put under general anesthesia of 50 mg/kg sodium pentobarbital through intraperitoneal injection. Fifty micrograms of LPS dissolved in 10 µl cerebrospinal fluid was microinjected into the lateral ventricle in the rat brains in all lesion groups. Injection of LPS was delivered at a rate of 2 µl/1 min (total 5 min) and injection needles were left in place for an additional 5 min.
10
LPS-Induced Bone Inflammation in Mice
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All animal procedures complied with the National Institutes of Health in the United States guidelines and were reviewed and approved by the local Hospital Animal Care and Use Committee (Institutional Animal Care and Use Committee approval number 2017070401, approval date: 7 September 2017). Ten-week-old male C57BL/6 mice were purchased from BioLasco Biotechnology (Taipei, Taiwan). Animals were initially anesthetized using an intraperitoneal injection of a 0.01 mL/kg mixture (1:1 v/v) of tiletamine-zolazepam (Zoletil® 50, Carros, France) and xylazine hydrochloride (Rompun® Bayer HealthCare, Berlin, Germany) and subsequently subjected to intrafemoral injections of 10 mg/kg LPS (Escherichia coli O127:B8, Sigma-Aldrich, St Louis, MO, USA) or vehicle PBS solution. On postinjection days 7 to 10, mice were euthanized (5 to 6 mice per group). The inoculated femur was immediately fixated in 10% formaldehyde and subjected to microcomputed tomography (micro-CT) analysis.
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