For BrdU labeling, mice were injected I.P. with 1 mg BrdU daily for six days. One day later, tissues were harvested and BrdU incorporation in iNKT cells was evaluated with the BrdU Flow Kit (BD Pharmingen) per the manufacturer’s instructions. As a cytokine-driven model of iNKT cell activation, mice were injected I.V. with 2mg ultra-pure LPS from E. coli O111:B4 (InvivoGen) per kg body weight. As an antigen-driven model of iNKT cell activation, mice were injected I.P. with 1 μg αGalCer analog KRN7000 (Avanti Polar Lipids). Both αGalCer and LPS were prepared in DMSO and diluted 1:10 (v/v) in sterile saline immediately prior to injection. Mice were sacrificed and tissues harvested after either 4 or 72 hours. In some experiments, mice were injected I.P. with 200μg ultra-low endotoxin azide-free anti-IFNγ (XMG1.2) or isotype control antibodies (Biolegend) 8 hours prior to αGalCer injection. For in vivo analysis of apoptosis mice were injected I.V. with the fluorescent poly-caspase binding reagent FAM-FLIVO (Immunochemistry Technologies) and analyzed per the manufacturer’s instructions. For ex-vivo intracellular cytokine staining, suspensions of SVF cells were cultured for two hours at 37°C in complete media in the presence of Brefeldin A prior to staining with flow cytometry antibodies.
Ultrapure lps from e coli o111 b4
Manufactured by InvivoGen
Sourced in United States
Ultrapure LPS (from E. coli O111:B4) is a laboratory product that provides a highly purified form of lipopolysaccharide (LPS) derived from the E. coli O111:B4 strain. LPS is a major component of the outer membrane of Gram-negative bacteria and is commonly used in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti caspase 1 d7f10, by Cell Signaling Technology (2 mentions)
Anti caspase 5 d3g4w, by Cell Signaling Technology (2 mentions)
Anti gsdmd g7422 from, by Merck Group (2 mentions)
Anti gsdmd n term e7h9g, by Cell Signaling Technology (2 mentions)
Anti il 1β mab601, by R&D Systems (2 mentions)
Anti actin c4 from, by MP Biomedicals (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Brdu flow kit, by BD (1 mentions)
Fam flivo, by ImmunoChemistry Technologies (1 mentions)
Krn7000, by Avanti Polar Lipids (1 mentions)
Ultrapure lps, by InvivoGen (1 mentions)
Recombinant mouse il 33, by BioLegend (1 mentions)
Recombinant mouse il 18, by BioLegend (1 mentions)
Papain, by Merck Group (1 mentions)
Dihydrorhodamine 123 dhr123, by Merck Group (1 mentions)
Recombinant human granulocyte macrophage colony stimulating factor rhgm csf, by Miltenyi Biotec (1 mentions)
Actinomycin d actd, by Merck Group (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
7 protocols using ultrapure lps from e coli o111 b4
1
Evaluating iNKT Cell Activation and Apoptosis
2
Lipopolysaccharide-Induced Kidney and Spleen Response
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Eight-week-old male C57BL/6 mice were i.p. administered either 200 μg of lipopolysaccharide (Ultrapure LPS; 1 mg/ml in sterile LPS-free phosphate-buffered saline, n = 6) in a total volume of 200 μl or an equal volume of sterile LPS-free phosphate-buffered saline (n = 6) (Ultrapure LPS from E. coli O111:B4, InvivoGen, Toulouse, France). Urine samples were collected and analyzed 24 hours post injection and immediately prior to sacrifice. Kidneys and spleens were then removed for morphological and protein studies.
3
HDM Sensitization and Challenge Protocol
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For preparation of HDM immunizations, whole crushed D. pteronyssinus HDM powder (Greer Laboratories Inc.) was resuspended in sterile PBS. For experiments that performed allergic sensitization and challenge, mice were anesthetized with isofluorane and administered HDM via the oropharynx at a concentration containing 23 μg Der p 1 protein in a volume of 40 μl during the primary sensitization. Beginning at 10 d after sensitization, mice were anesthetized with isofluorane and instilled with HDM in the oropharynx at a concentration containing 5.75 μg Der p 1 protein daily for 5 d during the allergic challenge phase.
For acute respiratory challenge experiments with HDM, papain, LPS, and recombinant cytokines, mice were anesthetized with isofluorane and administered the target molecules in a total volume of 40 μl diluted in sterile PBS via the oropharynx for the indicated days. On each day, mice were given HDM normalized to 5.75 μg Der p 1 protein (Greer Laboratories Inc.), 25 μg papain (from papaya latex, aseptically filled; Sigma-Aldrich), 10 μg Ultrapure LPS from E. coli O111:B4 (InvivoGen), 50 ng recombinant mouse IL-33 (BioLegend), and/or 500 ng recombinant mouse IL-18 (BioLegend) as described in the relevant figures.
For acute respiratory challenge experiments with HDM, papain, LPS, and recombinant cytokines, mice were anesthetized with isofluorane and administered the target molecules in a total volume of 40 μl diluted in sterile PBS via the oropharynx for the indicated days. On each day, mice were given HDM normalized to 5.75 μg Der p 1 protein (Greer Laboratories Inc.), 25 μg papain (from papaya latex, aseptically filled; Sigma-Aldrich), 10 μg Ultrapure LPS from E. coli O111:B4 (InvivoGen), 50 ng recombinant mouse IL-33 (BioLegend), and/or 500 ng recombinant mouse IL-18 (BioLegend) as described in the relevant figures.
4
Neutrophil Oxidative Burst Assay
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Ly6G+ CD11b+ cells in bone marrow were sorted as neutrophils using FACSAriaIII. Purified neutrophils were stimulated in the presence or absence of 100 ng/ml Ultra-pure LPS from E. coli O111:B4 (InvivoGen, San Diego, CA) or 10 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) (Kirin Brewery, Gunma, Japan) for 6 h, and then incubated with 10 μM of dihydrorhodamine-123 (DHR-123) (Sigma–Aldrich) for 30 min in quadruple cultures. Fluorescence intensity was determined using FACSCalibur. Results were calculated according to the following formula: rhodamin-123 fluorescence = mean fluorescence intensity (MFI) of stimulated cells/MFI of unstimulated cells × 100.
5
Immune Response Activation Assay
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Synthetic M-triDAP, ultrapure LPS from E.coli O111:B4 and synthetic polyriboinosinic:polyribocytidilic acid (poly-I:C) were from In vivogen (San Diego, CA). LPS was confirmed to be free of NOD1/NOD2 agonists using a test system described earlier (29 (link)). All agonists were dissolved in endotoxin-free water (In vivogen). Complexes of poly-I:C with Lipofectamine 3000 (lipo-poly-I:C) were prepared as recommended by the manufacturer of Lipofectamine (Thermo Fisher Scientific, Waltham, MA). Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) was from Miltenyi Biotec (Bergisch Gladbach, Germany), and recombinant human interferon-beta (rhIFN-β1b) from Generium (Moscow, Russia). Complete culture medium (CCM) was RPMI-1640 supplemented with 2-mM L-glutamine and 10% fetal calf serum (all from Thermo Fisher Scientific). IκB kinase β inhibitor (PF-184) was from Tocris (Bristol, UK), actinomycin D (ActD) from Sigma (St-Louis, MO).
6
Apoptosis and Pyroptosis Pathway Analysis
7
Inflammasome Signaling Pathway Analysis
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The following antibodies were used: anti-Caspase-1 (D7F10 from Cell Signaling Technology, Danvers, MA, USA), anti-Caspase-4 (4450 from Cell Signaling Technology, Danvers, MA, USA), anti-Caspase-5 (D3G4W from Cell Signaling Technology, Danvers, MA, USA); anti-actin (C4 from MP Biomedicals); anti-GSDMD (G7422 from Sigma-Aldrich, St. Louis, MO, USA), anti-GSDMD N-term (E7H9G from Cell Signaling Technology, Danvers, MA, USA), anti-IL-1β (MAB601, R&D Systems, Inc., Minneapolis, MN, USA), anti-IL-1β cleaved, Asp116 (PA5-105048 from Thermo Fisher, Waltham, MA, USA). All cell culture media reagents were purchased from Thermo Fisher (Waltham, MA, USA). Ultrapure LPS (from E. coli O111:B4) was purchased from Invivogen (San Diego, CA, USA). Unless otherwise indicated, all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).
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