Tanshinone I (Tan I, HY-N0134), MCC950 (HY-12815 A), and nigericin (HY-127019) were from MedChemExpress. ATP, SiO2, Pam3CSK4, poly (I:C), poly (dA:dT), and ultrapure lipopolysaccharide (LPS) were from Invivogen. Phorbol 12-myristate 13-acetate (PMA, P8139) and DMSO (D2650) were from Sigma Aldrich. The Starfect high-efficiency transfection reagent (C101-10) was from GenStar. Antibodies were used as follows: anti-mouse IL-1β (1:1000, AF-401-NA, R&D), anti-mouse caspase-1 (1:1000, AG-20B-0042, Adipogen), anti-human cleaved IL-1β (1:2000, 12242, Cell Signaling Technology), anti-human caspase-1 (1:2000, 4199 S, Cell Signaling Technology), anti-NLRP3 (1:2000, AG-20B-0014, Adipogen), anti-ASC (1:1000, sc-22514-R, Santa Cruz), anti-Flag (1:2000, 20543-1-AP, Proteintech), and anti-GAPDH (1:5000, 60004-1-Ig, Proteintech).
Ultrapure lipopolysaccharide lps
Manufactured by InvivoGen
Sourced in United States
Ultrapure lipopolysaccharide (LPS) is a laboratory product manufactured by InvivoGen. It is a highly purified form of LPS, a major component of the outer membrane of Gram-negative bacteria. The core function of this product is to serve as a tool for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Pam3csk4, by InvivoGen (3 mentions)
Adenosine triphosphate (atp), by Merck Group (3 mentions)
Anti mouse il 1β, by R&D Systems (2 mentions)
Anti mouse caspase 1, by Adipogen (2 mentions)
Af 401 na, by R&D Systems (2 mentions)
Ag 20b 0042, by Adipogen (2 mentions)
Ag 20b 0014, by Adipogen (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Mannan, by Merck Group (2 mentions)
Mitosox, by Thermo Fisher Scientific (2 mentions)
Dmso d2650, by Merck Group (1 mentions)
Sc 22514 r, by Santa Cruz Biotechnology (1 mentions)
Nigericin hy 127019, by MedChemExpress (1 mentions)
Anti human cleaved il 1β, by Cell Signaling Technology (1 mentions)
Anti human caspase 1, by Cell Signaling Technology (1 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (1 mentions)
Z vad fmk, by Apexbio (1 mentions)
Recombinant tnf α, by R&D Systems (1 mentions)
Mcc950, by InvivoGen (1 mentions)
Streptomycin, by Merck Group (1 mentions)
15 protocols using ultrapure lipopolysaccharide lps
1
Inflammasome Activation Reagents Protocol
2
Inflammasome-mediated Immune Responses
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Isolated neutrophils and macrophages were stimulated with ultrapure lipopolysaccharide (LPS) (Invivogen) at 500 ng/mL for 3 h, and 3 mM ATP (Sigma-Aldrich) was added for 1 h to selectively activate NLRP3. In some experiments, 20 ng/mL GM-CSF was also included in the cultures, which may affect P2X7 functional expression. To induce caspase-3 and GSDME activation, 100 ng/mL recombinant TNFα (R&D Systems) with 2 µM Birinapant Smac mimetic (MedChemExpress) or 2.5 ng/mL cycloheximide (final concentrations) were added for 4 h. The pan caspase inhibitor Z-VAD-FMK (APExBIO), the NLRP3 inhibitor MCC950 (Invivogen) and the ROS inhibitor diphenyleneiodonium chloride (DPI, Sigma) were dissolved in dimethyl sulfoxide (DMSO) (Thermo Fisher Scientific). Cells were pretreated with ZVAD at 50 µM for 30 min, with MCC920 at 2 µM for 40 min, with DPI at 10 µM for 20 min, or with the equivalent volume of DMSO as vehicle control. Cells were incubated with Pseudomonas aeruginosa strains at an MOI = 30 for 25 min for western blots or 1 h for cytokine production and LDH release. Neutrophils were suspended in media containing 20 ng/mL GM-CSF to reduce spontaneous activation of caspase-3. Macrophages were pretreated with 5 mM glycine for 30 min to inhibit macrophage pyroptosis as described25 (link),26 (link).
3
Whole Blood Cytokine Assay with Immune Stimuli
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Heparinized venous blood was processed for culture within 6 hours after venipuncture. Whole blood was diluted 2 times in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 100 U/mL penicillin (Astellas Pharma BV, Leiden, the Netherlands), 10 µg/mL streptomycin (Sigma-Aldrich, Saint Louis, MO, USA), 1 mM pyruvate (Sigma-Aldrich) and 2 mM L-glutamine (Sigma-Aldrich) and stimulated with 50 µg/mL polyinosinic-polycytidylic acid high molecular weight (poly(I:C); InvivoGen, San Diego, CA, USA), 50 ng/mL Pam2CGDPKHPKSF (FSL-1; InvivoGen), 100 ng/mL Pam3CSK4 (Pam3; EMC Microcollections GmbH, Tübingen, Germany), 100 ng/mL ultrapure lipopolysaccharide (LPS; InvivoGen), 10 µg/mL γ-D-Glu-mDAP (iE-DAP; InvivoGen), 100 µg/mL mannan (Sigma-Aldrich), 100 µg/mL curdlan (Wako Chemicals GmbH, Neuss, Germany) or 5 ng/mL 1-(palmitoyl)-2-(5-keto-6-octene-dioyl)phosphatidylcholine (KOdiA-PC; Cayman Chemicals, Ann Arbor, MI, USA), alone or in combination (Table 1 ). Medium was used as a negative control and 2 µg/mL phytohemagglutinin (PHA; Remel, Dartford, UK), a mitogen, as a positive control. 100 µL of ligand(s) in medium was added to wells containing 100 µL of diluted blood in 96-well round-bottom plates (Nunc; Roskilde, Denmark) and incubated in the presence of 5% CO2 at 37°C for 24 hours. Supernatants were harvested and stored at −80°C.
4
Monocyte Cytokine Response to Fungal Infection
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Human monocytes were positively selected by using CD14 microbeads coupled to a magnetic cell sorting system (Miltenyi Biotec). The isolated cells were incubated in a 24-well plate at 37 °C and 5% CO2, and stimulated for three hours with specific inhibitors reported in previous studies: 5 µM Interleukin-1 Receptor-Associated Kinase (IRAK) inhibitor (CAS 509093-47-4, Merck), 5 µM Spleen tyrosine kinase (Syk) inhibitor (ER 27319 maleate, R&D systems), 10 µM NF-KB inhibitor (Bay11-7082, In vivogen), 10 µg/mL human TLR2 Neutralizing antibody (Ab) (PAb-hTLR2, In vivogen), and 5 µM TLR4 inhibitor (CLI-095, In vivogen) (35 (link)–38 (link)). Inhibition of IRAK, Syk, and the nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB) was performed at a density of 1x106 cells/well in 1 mL of RPMI medium supplemented with 10% v/v FBS, while TLRs at a density of 5x105 cells/well in 0,5 mL of total volume. Subsequently, the monocytes were infected with the mucoralean fungi at MOI 5 for sixteen hours. Then, the supernatants were collected and the secreted cytokines were determined by ELISA MAX™ Deluxe kit (Biolegend). PAM3CSKA (In vivoGen) and ultrapure Lipopolysaccharide (LPS) (In vivoGen) were used as agonists for TLR axis activation, while Mannan (Sigma Aldrich) was used for CTL activation.
5
Recombinant TSP1 Domains Characterization
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Adenosine 5-triphosphate disodium salt hydrate (ATP) and phorbol 12-myristate 13-acetate (PMA) were from Sigma. RPMI 1640 Medium, penicillin-streptomycin (Pen-Strep), L-glutamine (L-Gln), Versene and Opti-MEM I were from Life Technologies. Ultrapure lipopolysaccharide (LPS) and Pam3CSK4 were from InvivoGen. Fetal bovine serum (FBS) was from Gemini BioProducts. Human TSP1 was purified from platelets obtained from the National Institutes of Health Department of Transfusion Medicine, as previously described69 . Recombinant TSP1 domains comprising the type 1 repeats (3TSR), N-terminal, oligomerization and von Willebrand C domains (NoC1), and EGF repeats, calcium-binding repeats and C-terminal domain (E123CaG1) were kind gifts from Dr. Jack Lawler70 (link) and Dr. Dean Mosher71 (link)72 (link), respectively.
6
Inflammasome Activation Assay Protocol
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ATP, phorbol myristate acetate, cytochalasin D and BAPTA-AM were from Sigma-Aldrich. Ultrapure lipopolysaccharide (LPS) was from Invivogen. DPI was from Calbiochem. Lipofectamine 2000 and MitoSOX were from Life Technologies. Calcium-free and calcium-containing Dulbecco's modified Eagle's medium (DMEM) were from US Biologicals. Caspase-1 inhibitor Z-YVAD-FMK and caspase-3 inhibitor Z-DEVD-FMK were from Enzo Life Sciences. The lactate dehydrogenase assay kit and in situ cell death detection kit (TUNEL) were from Roche. FLICA capase-1 kit was from Immunochemistry. Antibodies used for immunoblotting were as follows: anti-mouse caspase-1 (AG-20B-0042-C100, Adipogen, 1 μg ml−1), anti-mouse β-actin (sc-1615 horseradish peroxidase (HRP), Santa Cruz Biotechnology, 0.1 μg ml−1) and anti-mouse IL-1β (AF-401-NA, R&D Systems, 0.25 μg ml−1). Imject Alum and streptavidin-HRP were from Pierce.
7
Macrophage Differentiation and NLRP3 Inflammasome
8
Antioxidant and Anti-inflammatory Compounds
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Carnosol (from Rosemarinus officinalis) and curcumin (from Curcuma longa) were purchased from Sigma-Aldrich and dissolved in DMSO. Human hemoglobin was purchased from Sigma-Aldrich and dissolved in RPMI. Ultrapure lipopolysaccharide (LPS) was purchased from Invivogen. The HO-1 inhibitor tin-protoporphyrin IX (SnPP) was purchased from Frontier Scientific and dissolved in 50 mM TRIS buffer.
9
Isolation of Peritoneal Macrophages from Mice
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Peritoneal macrophages were isolated from Nlrp3−/− and PXR−/− mice and their littermate wild-type counterparts (male, 8–10 weeks of age; all bred in house) 48 hours after receiving an i.p. injection of 4% thioglycolate-injected mice (BD Biosciences, San Jose, CA), as we have done previously (Ng et al., 2010 (link); Hirota et al., 2011 (link)). Isolated macrophages were plated in complete RPMI media at 5 × 105 cells/well of a 24-well plate overnight and stimulated with 100 ng/ml ultra-pure lipopolysaccharide (LPS; Invivogen) in serum-free Opti-MEM for 30 minutes before challenge. For experiments with knockout mice, littermates were used as the wild-type control group for all experiments to control for potential microbiota-dependent differences in phenotype. All studies were approved by the University of Calgary’s Health Sciences Animal Care Committee (protocol AC15-0181). All approved activities conform to the guidelines and regulation for laboratory animal use set forth by the Canadian Council for Animal Care.
10
Investigating Macrophage Activation Pathways
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Ultrapure lipopolysaccharide (LPS) and the PI3K inhibitor, LY294002, were from Invivogen (Toulouse, France). The Syk inhibitor, R788, was from AdooQ BioScience (Irvine, CA, USA). Recombinant human M-CSF was from PeproTech (Rocky Hill, NJ, USA). Recombinant human IL-4 and GM-CSF were from Immunotools (Friesoythe, Germany). Lymphoprep was from Stemcell Technologies (Grenoble, France). Primary antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). The Syk inhibitor, piceatannol, methyl-β-cyclodextrin (M-βCD), secondary antibodies, cell culture reagents and all other chemicals were from Sigma Aldrich (St. Louis, MO, USA). Human FcR binding inhibitor was from eBioscience. BCP crystals were synthesized by alkaline hydrolysis of brushite as described previously [28 (link)] and contain partially carbonate-substituted hydroxyapatite in addition to octacalcium phosphate. OA synovial fluid was obtained with permission from The Mater Misericordiae Hospital Research Ethics Committee.
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