On PN150, 200 μg Escherichia coli lipopolysaccharide (O55:B5, LPS Ultrapure, InvivoGen, Toulouse, France) (19 (link), 20 (link)) suspended in saline solution to a total volume of 200 μl or an equivalent volume of saline (SAL) was instilled intratracheally in both Control and Obese animals, resulting in four experimental groups (n = 9, each): (1) Control-SAL; (2) Control-ALI ; (3) Obese-SAL; and (4) Obese-ALI . After 24 h, echocardiography and lung mechanics were analyzed, and biological samples were collected. Lungs were stored for further evaluation of histology, enzyme-linked immunosorbent assay (ELISA), and myeloperoxidase (MPO) activity. Gene expression was also quantified in macrophages and neutrophils obtained from blood and BALF.
Lps ultra pure
Manufactured by InvivoGen
Sourced in France, United States
LPS Ultra pure is a high-purity lipopolysaccharide (LPS) product. It is a component derived from the outer membrane of Gram-negative bacteria. The product is designed to provide a reliable and consistent source of LPS for research applications.
Automatically generated - may contain errors
Lab products found in correlation
Pam3csk4, by InvivoGen (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Pyruvate, by Thermo Fisher Scientific (2 mentions)
Rgm csf, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 dutch modification, by Thermo Fisher Scientific (2 mentions)
β mercaptoethanol, by Merck Group (2 mentions)
Fetal calf serum biowhittaker, by Lonza (2 mentions)
Tlrl 1826, by InvivoGen (2 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Paramagnetic cd14 beads, by Miltenyi Biotec (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Bicoll seperation, by Harvard Bioscience (1 mentions)
Refobacin, by Merck Group (1 mentions)
Gm csf, by Bayer (1 mentions)
Interleukin 4 (il 4), by Miltenyi Biotec (1 mentions)
P38 mapk, by Cell Signaling Technology (1 mentions)
Insulin, by Thermo Fisher Scientific (1 mentions)
Redd1, by Proteintech (1 mentions)
11 protocols using lps ultra pure
1
Obese Animal Model of ALI
2
Generating Monocyte-Derived Dendritic Cells
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DCs were generated from PBMCs as previously described24 (link). Briefly, PBMCs were isolated from healthy volunteers by ficoll (Bicoll Seperation, Biochrom AG) density gradient centrifugation. Magnetic activated cell sorting with paramagnetic CD14-beads (Miltenyi Biotec) was used to further separate monocytes. Monocyte-derived dendritic cells (DCs) were generated in RPMI-1640 supplemented with 10% FBS (Sigma-Aldrich), 120 mg/l Refobacin (Merck), 10 ng/ml IL-4 (Miltenyi Biotec) and 100 ng/ml GM-CSF (Bayer Healthcare) for 5 to 6 days.
Co-cultivation experiments of DCs with A. fumigatus or C. albicans and E.coli were performed on day 6 with a multiplicity of infection (MOI) of 1 in a concentration of 1*106/ml. LPS (1 μg/ml, Sigma-Aldrich), LPS ultra-pure (1 μg/ml), zymosan depleted (100 μg/ml), Pam3CSK4 (100 ng/ml) (Invivogen) were used in the indicated concentrations.
Co-cultivation experiments of DCs with A. fumigatus or C. albicans and E.coli were performed on day 6 with a multiplicity of infection (MOI) of 1 in a concentration of 1*106/ml. LPS (1 μg/ml, Sigma-Aldrich), LPS ultra-pure (1 μg/ml), zymosan depleted (100 μg/ml), Pam3CSK4 (100 ng/ml) (Invivogen) were used in the indicated concentrations.
3
Inflammatory Signaling Pathway Assays
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Insulin was obtained from Life Technologies (Saint Aubin, France), LPS ultrapure from InvivoGen (San Diego, USA) and IL-1β from PeproTech France (Neuilly sur seine, France). Antibodies were obtained from the following companies: REDD1 from Proteintech (Chicago, IL); pT180/Y182 p38 MAPK, p38 MAPK, JNK, pT183/Y185 JNK, pS536p65 NF-κB, IL1β, pT308PKB, PKB, ERK from Cell Signaling Technology (Beverly, MA); NFκB, HSP90 from Santa Cruz Biotechnology (Heidelberg, Germany); tubulin from Sigma-Aldrich; NLRP3, caspase-1 (p20) from Adipogen (San Diego, USA). The primer sets for real time PCR were purchased from Eurogentec (Seraing, Belgium) and Qiagen (Courtaboeuf, France). Culture media were obtained from Life Technologies (Saint Aubin, France). Rapamycin was obtained from Calbiochem (Nottingham, UK).
4
Characterization of Bacterial Lipid Mediators
5
Dendritic Cell Culture and Stimulation
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DC were cultured from bone marrow obtained from wild-type (WT) C57Bl6, Sdc-1-/- and Balb/c mice according to a method adopted from Lutz et al. [27 (link), 28 (link)]. In short, femora and tibiae were harvested after cervical dislocation and bone marrow was flushed with medium (RPMI-1640 Dutch modification (Invitrogen, USA), supplemented with 50 μM β-mercaptoethanol (Sigma-Aldrich, St Louise, USA), 1% glutamax (Invitrogen), 1% pyruvate (Invitrogen) and 10,000 U/ml penicillin-streptomycin (Invitrogen). Cells were suspended in medium with 10% fetal calf serum (BioWhittaker, Lonza, Basel, Switzerland), supplemented with 20 ng/ml rGM-CSF (PeproTech, Rocky Hill, USA) and subsequently cultured for 9 days in six well plates (0.8 x 106 cells/well, Corning Incorporated, USA) at 37˚C and 5% CO2. At day 3 and day 6 medium was refreshed. After 8 days of culture, DC were stimulated with different TLR ligands for 24 hours, i.e for TLR2 stimulation 1 μg/ml PAM3CysSerLys4 (PAM3, tlrl-pms, InvivoGen), for TLR4 stimulation 1 μg/ml LPS Ultra pure (InvivoGen, San Diego, USA), and for TLR9 stimulation 5 μg/ml ODN1826 (tlrl-1826, InvivoGen). Unstimulated DC received no additives. At day nine, cells were harvested. Culture supernatant was collected and stored at -20°C for cytokine measurement. Cells were analyzed by flow cytometry or used in co-culture with T cells.
6
Inflammasome Activation and Cytokine Detection
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Cell culture reagents (e.g., PBS, DMEM, RPMI, Trypsin‐EDTA) were from Gibco, Thermo Fisher Scientific. Stimuli were as follow: LPS (UltraPure, Invivogen), nigericin (Thermo Fisher Scientific), PFO (Biotrend), LFn‐BsaK, LFn‐MxiH and PA were produced in‐house as described previously (19), and uric acid used for in‐house production of MSU crystals (Sigma‐Aldrich). Inhibitors used were CRID3 (Tocris) and VX‐765 (Selleckchem). Antibodies were as follow: anti‐ASC polyclonal antibody (AL177, Adipogen, 50 µg ml−1) or anti‐ASC monoclonal antibody (N‐15, Santa Cruz, 1:500), anti‐Caspase‐1 p20 antibody (Casper‐1, Adipogen, 1:1,000), anti‐IL‐1β antibody (BAF401, R&D Systems, 1:1,000), and anti‐GSDMD (L60, Cell Signaling Technology, 1:1,000). DRAQ5 was from Thermo Fisher Scientific. For FACS, Abs used were anti‐CD45‐BV421, Ly6G‐FITC and Ly6C‐PerCP (BD Biosciences, 1:100). HTRF for human or mouse IL‐1β were from CisBio. ELISA kits were used to detect IL‐1β, TNFα, IL‐6 or CXCL1 (DuoSet, R&D Systems). Cell viability was measured with the Cell Titer Blue kit (CTB, Promega).
7
Monocyte Transfection and TLR Stimulation
8
Dendritic Cell Culture from Murine Bone Marrow
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Dendritic cell culture DC were cultured from bone marrow obtained from wild-type (WT) C57Bl6, Sdc-1 -/-and Balb/c mice according to a method adopted from Lutz et al. (24, 25) . In short, femora and tibiae were harvested after cervical dislocation and bone marrow was flushed with medium (RPMI-1640 Dutch modification (Invitrogen, USA), supplemented with 50 µM β-mercaptoethanol (Sigma-Aldrich, St Louise, USA), 1% glutamax (Invitrogen), 1% pyruvate (Invitrogen) and 10,000 U/ml penicillin-streptomycin (Invitrogen). Cells were suspended in medium with 10% fetal calf serum (BioWhittaker, Lonza, Basel, Switzerland), supplemented with 20 ng/ml rGM-CSF (PeproTech, Rocky Hill, USA) and subsequently cultured for 9 days in six well plates (0.8 x 10 6 cells/well, Corning Incorporated, USA) at 37˚C and 5% CO 2 . At day 3 and day 6 medium was refreshed. After 8 days of culture, DC were stimulated with different TLR ligands for 24 hours, i.e for TLR2 stimulation 1 µg/ml PAM 3 CysSerLys 4 (PAM 3 , tlrl-pms, InvivoGen), for TLR4 stimulation 1 µg/ml LPS Ultra pure (InvivoGen, San Diego, USA), and for TLR9 stimulation 5 µg/ml ODN1826 (tlrl-1826, InvivoGen). Unstimulated DC received no additives. At day nine, cells were harvested. Culture supernatant was collected and stored at -20°C for cytokine measurement. Cells were analyzed by flow cytometry or used in co-culture with splenocytes.
9
Lymphatic cell cytokine production
10
Macrophage Stimulation Assay
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Stimulation experiments were performed on adherent peritoneal macrophages which were primed by 100 ng ml -1 LPS Ultrapure (Invivogen, France) for 4 h before been stimulated by purified proteins for 20 h.
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