CLAMS chambers (Columbus Instruments) housed in a temperature controlled environmental chamber were used to quantify energy expenditure. Oxygen consumption rate (VO2), CO2 release rate (VCO2), food intake, and activity were recorded after 1–2-day acclimation in the CLAMS chamber. Conscious mice were administered TLR ligands at indicated doses. Pam3CSK4 (1 and 10 mg/kg, i.p.), Flagellin from S. typhimurium (0.5 mg/kg, i.p.), Imiquimod R837 (10 mg/kg, i.p.), ODN1826 (2.5 mg/kg, i.p.), ODN1585 (5 mg/kg, i.p.), Poly (I:C) LMW (40 mg/kg, i.p.) and HKLM (2.5 × 109 per mouse, i.p.) were purchased from Invivogen. PGN from Bacillus subtilis (69554) (2.5, 5, and 10 mg/kg, i.p.) was purchased from Sigma Aldrich. LPS (from E. coli serotype O111:B4, catalogue # L3024–25 mg) was purchased from Sigma Aldrich, reconstituted in sterile saline at 5 mg/ml, and stored in 100 µl aliquots at −20°C. During the course of our experiments, we used 5 different lots of LPS and observed small variations in the potency of LPS amongst these 5 lots. Thus, to standardize each lot, a dose response curve for suppression of VO2 was performed in C57BL6/J female mice housed at 22°C. Dose of LPS that significantly suppressed metabolic rate (ranging from 1–1.5 mg/kg) was selected for the subsequent experiments.
Lps from e coli serotype o111 b4
Manufactured by Merck Group
Sourced in Germany, United States
LPS) from E. coli serotype O111:B4 is a laboratory reagent used for research purposes. It is a lipopolysaccharide extracted from the cell walls of Escherichia coli bacteria of the O111:B4 serotype. This product is commonly used in scientific research to study the biological effects of endotoxins.
Automatically generated - may contain errors
Lab products found in correlation
Poly 1 c lmw, by InvivoGen (1 mentions)
Imiquimod r837, by InvivoGen (1 mentions)
Odn1585, by InvivoGen (1 mentions)
Clams chambers, by Columbus Instruments (1 mentions)
Odn1826, by InvivoGen (1 mentions)
Pam3csk4, by InvivoGen (1 mentions)
Sodium butyrate, by Merck Group (1 mentions)
Il 1β, by Thermo Fisher Scientific (1 mentions)
Ms 275, by Selleck Chemicals (1 mentions)
Entinostat, by Selleck Chemicals (1 mentions)
Flunarizine dihydrochloride, by Merck Group (1 mentions)
N acetyl l cysteine nac, by Merck Group (1 mentions)
Potassium chloride (kcl), by Merck Group (1 mentions)
Z vad fmk, by InvivoGen (1 mentions)
Mcc950, by InvivoGen (1 mentions)
15 deoxy δ12 14 prostaglandin j2, by Cayman Chemical (1 mentions)
6 protocols using lps from e coli serotype o111 b4
1
Measuring Metabolic Responses to Immune Stimuli
2
HDAC Inhibition Modulates Immune Response
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Cells or biopsies were pretreated with suberoylanilide hydroxamid acid (SAHA; Vorinostat) (InvivoGen, USA) MS-275 (Entinostat; Selleckchem, USA) or sodium-butyrate (SB) (Sigma-Aldrich, Germany) for 2 hrs prior to the start of the stimulation with either heat-inactivated E. coli Nissle 1917 (Ardeypharm, Germany), IL1β (PeproTech, Germany) or LPS from E. coli serotype O111:B4 (Sigma-Aldrich, Germany) which took place in parallel to HDAC inhibition for additional 18 hrs adding up to a total of 20 hrs of treatment. Treatment with the NF-κB inhibitor Helenalin (Enzo Life Sciences, USA) took place for 1 h right at the beginning of the 20 hrs and was then removed to avoid cytotoxic effects.
3
Flunarizine Protects Mice from LPS-Induced Sepsis
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All experiments were approved by the local authorities at Hannover Medical School and conducted in accordance with institutional and governmental guidelines (LAVES Lower Saxony, Ref Nr. 12/0681). Eight to 12 weeks old C57BL/6 J male mice were purchased from Charles River (Sulzfeld, Germany) and the Central Animal Facility of Hanover Medical School. For 3 days the mice received 25 mg/kg bodyweight (bw) of Flunarizine dihydrochloride (Sigma-Aldrich, St. Louis, MO) or a vehicle control orally once a day. On the third day, 17.5 mg/kg bw lipopolysaccharide (LPS) from E. coli serotype O111:B4 (Sigma-Aldrich, St. Louis, MO) was administered intraperitoneally (i.p.). After 12 hrs the mice were sacrificied for organ harvest and further molecular analysis. For survival studies, mice received 25 mg/kg bw Flunarizine or vehicle control orally for three days and were injected with 20 mg/kg bw LPS on the third day.
4
Modulating Macrophage Inflammasome Activation
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We added CPPD (InvivoGen), mycolactone, M. ulcerans or vesicles, at various concentrations, with or without KCl (Sigma P5405) to inhibit K+ channels; MCC950 (InvivoGen inh_mcc), an NLRP3 inhibitor; 15-deoxyΔ12,14-prostaglandin J2 (Cayman Chemicals ref. 18570), an NLRP3/1 inhibitor; N-acetyl-L-cysteine (NAC, Sigma A9165), a ROS inhibitor; and Z-VAD-FMK (InvivoGen tlrl_vad), a pan-caspase inhibitor. The macrophages were then incubated for 24 h with or without LPS (from E. coli serotype O111:B4; Sigma-Aldrich) added to a final concentration of 50 ng/mL.
5
LPS-Induced Inflammatory Response Modulation
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Cells (1x105 cells/well) were activated with 10μL of lipopolysaccharide (LPS) from E. coli serotype O111:B4 (Sigma, St. Louis, MI, USA) at 500 ng/ml. At the same time, aliquots of BRP (40–100 μg/ml) were added to each well and the plates were incubated for 48 hours at 37°C in 5% CO2 with LPS and BRP or controls. Cells added with the vehicle (DMSO) with and without LPS and/or BRP were used as controls [26 (link)].
6
Generating Lipid Bilayer Voids and Backfilling
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LPS from E. coli serotype O111:B4 (phenol extract), purchased from Sigma-Aldrich (St. Louis, MO), was used to disrupt membranes as previously described25 (link). To generate voids in lipid bilayers, DOPC SLBs were incubated with LPS at concentrations of 100-500 μg/ mL in PBS for 20 min and then washed with fresh PBS. Subsequent to characterisation by microscopy, various secondary components were used for backfilling. Globular proteins were used in preliminary backfilling experiments (see Supplementary Fig. S2 ), by incubating with 0.5 mg/mL bovine serum albumin tagged with an AlexaFluor label (AF594 or AF647) in PBS, followed by washing with fresh PBS. Backfilling with DOPC or DPSC liposomes was by incubation with 0.5 mg/mL liposomes for up to 20 min, followed by washing with PBS. DSPC liposomes were kept at 60 °C until use (above their phase transition temperature)29 . Adding hot DSPC liposomes to the room temperature DOPC SLB during backfilling allowed DSPC lipids to form a domain contiguous with the surrounding DOPC but complete mixing was prevented as the DSPC cools below its phase transition.
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