Polyinosinic-polycytidylic acid poly (I:C) was obtained from Sigma-Aldrich (St Louis, MO, USA) as the sodium salt. All reagents and solvents in LC-MS/MS analyses were ACS grade. Ammonium bicarbonate (ABC),2,2,2,-trifluoroethanol(TFE), and acetic acid were purchased from Sigma-Aldrich. Iodoacetamide (IDA), dithiothreitol (DTT), acetonitrile (ACN), formic acid, and methanol were purchased from Thermo Scientific (Waltham, MA, USA). Urea ultra was from MP Biomedicals (Santa Ana, CA, USA). Sequencing-grade modified trypsin and LysC were from Promega (Madison, WI, USA). CD140b (platelet-derived growth factor receptor beta [PDGFRB]) monoclonal antibody (APB5) was from Invitrogen (San Diego, CA, USA). Anti-alpha smooth muscle actin antibody (ab5694) was from Abcam (Cambridge, MA, USA). Goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa fluor 568 (A-11036) and goat anti-Rat IgG (H+L) cross-adsorbed secondary antibody, Alexa fluor 488 (A-11006) were from Invitrogen.
Polyinosinic polycytidylic acid poly 1 c
Manufactured by Merck Group
Sourced in United States, Germany
Polyinosinic:polycytidylic acid (poly(I:C)) is a synthetic double-stranded RNA molecule that is structurally similar to viral RNA. It is commonly used as a tool in biological research to induce innate immune responses in cells and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Acetic acid, by Merck Group (3 mentions)
Lipopolysaccharide lps, by Merck Group (3 mentions)
Pd98059, by Merck Group (3 mentions)
Anti mouse cd40 clone 1c10, by Southern Biotech (3 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Ammonium bicarbonate abc 2 2 2 trifluoroethanol tfe, by Merck Group (2 mentions)
Urea ultra, by MP Biomedicals (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Sb203580, by Merck Group (2 mentions)
Pam3csk4, by InvivoGen (2 mentions)
Ifn γ, by R&D Systems (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Mouse monoclonal anti c myc antibody, by Merck Group (2 mentions)
Mouse monoclonal anti flag m2 antibody, by Merck Group (2 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions)
Prrsv n protein antibody, by GeneTex (2 mentions)
Mouse anti β actin monoclonal antibody, by GenScript (2 mentions)
74 protocols using polyinosinic polycytidylic acid poly 1 c
1
Poly(I:C)-Based Proteomics Analysis
2
Generating Bone Marrow-Derived Dendritic Cells
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Bone marrow-derived DCs were generated from the femur and tibia of female 7-8 week old wild-type (wt) BALB/c mice (H-2Dd) (The Janvier Labs, France) by exposing the bone marrow and flushing out the cells with a sterile syringe. The harvested cells were cultured in IMDM supplemented with 10% heat inactivated FBS, 1% PeSt, 1% HEPES, 50 μM β-mercaptoethanol. Culture medium was supplemented with 20 ng/mL recombinant murine IL-4 and 20 ng/mL recombinant murine GM-CSF (Nordic BioSite, Stockholm, Sweden). Bone marrow cells were plated on non-treated Petri dishes (Sarstedt, Nümbrecht, Germany). Medium was replaced every 3 days. On day 7 the non-adherent imDCs were harvested and treated for 18 h with a cocktail of combined Toll-like receptor ligands with IFN-γ (COMBIG), consisting of 2.5 μg/mL R848 (InvivoGen, Sam Diego, CA), 20 μg/mL polyinosinic:polycytidylic acid (polyI:C) (Sigma-Aldrich, St. Louis, MO) and 1000 IU/mL IFN-γ (Nordic Biosite)22 (link) in order to obtain mature alloDCs. Ad5M(gp100)-alloDCs were obtained by transducing pelleted imDCs for 2 h, at 37oC with 2000 evg/cell Ad5M(gp100) and matured as above. Cells were cultured in a humidified incubator with a 5% CO2 atmosphere at 37°C.
3
Immunocytochemistry of TLR3 and TLR4
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MSC (5,000) were cultured in 250 μL of EM in 8-well Permanox chamber slides (Thermo Fisher Scientific) until 60–80% confluency. Cells were exposed to 1 μg/mL of polyinosinic:polycytidylic acid (poly I:C; Sigma-Aldrich) and 10 ng/mL of lipopolysaccharide (LPS; Sigma-Aldrich) for one hour to induce TLR3 and TLR4 expression. For ICC, cells were washed thrice with PBS, fixed in situ for 5 minutes with 4% paraformaldehyde (PFA; Invitrogen), washed thrice again with PBS and permeabilized with 0.1% Triton for 15 minutes. Cells were then washed again and treated with 3% hydrogen peroxide for 15 minutes. Non-specific antibody binding was blocked by incubation with 5% FBS for 10 minutes followed by one hour incubation with polyclonal primary antibody to TLR3 or TLR4 (both Imgenex, San Diego, CA). Cells were then washed thrice with PBS and incubated for one hour with goat anti-rabbit secondary antibody (Abcam). Bound antibody linked to horseradish peroxidase (HRP) was detected using 3,3'-diaminobenzidine chromogen (Dako, Burlington, ON). Cells were counterstained with Harris hematoxylin to visualize nuclei, and images were acquired on a phase contrast microscope.
4
TLR3 Activation Induces Th17 Polarization
5
Induction and Modulation of Liver Fibrosis
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For the induction of CCl4-mediated liver fibrosis, age-matched (8-week-old) male C57BL/6J mice received intraperitoneal injections of CCl4 (20% CCl4 olive oil solution, 2 ml/kg of animal body weight) three times per week for 2 weeks (Fig 1A ). Mice in the control group received an equal volume of olive oil (vehicle). Mice were sacrificed at 12 hours after the final injection. To generate cholestasis-induced liver fibrosis, age-matched (8-week-old) male C57BL/6J mice were underwent BDL [21 (link)]. Briefly, mice were anesthetized with isoflurane and BDL was performed after midline laparotomy. The common bile duct was ligated with 5–0 surgical silk and transected between the 2 distal ligations. Sham-operated mice had their common bile duct exposed and manipulated but not ligated or transected. For inhibition of ADH, 10 μg of 4-MP/g of animal body weight was injected intraperitoneally three times per week for 2 weeks (Fig 1A ). For the NK cell activity assay, 2.5 μg polyinosinic-polycytidylic acid (poly I:C)/g of animal body weight (Sigma Aldrich, St. Louis, MO) was injected 12 hours after a single 4-MP challenge [22 (link)].
6
Monocyte-Derived Dendritic Cell Protocol
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PBMCs were incubated in T-75 flasks (Corning) for 2 hours and nonadherent cells (mostly peripheral blood lymphocytes) were saved. Adherent cells (mostly monocytes) were washed three times with phosphate-buffered saline, differentiated over 6 days into immature DCs with granulocyte-macrophage colony-stimulating factor (Gentaur, Brussels, Belgium, 50 ng/ml) and IL-4 (Gentaur, 25 ng/ml). Medium was changed every 2 days. Immature DCs were then maturated for 24 hours in culture medium with IFN-γ (Shenandoah Biotechnology, Warwick, PA, 1,000 IU/ml), polyinosinic:polycytidylic acid (poly(I:C); Sigma-Aldrich, St Louis, MO, 20 µg/ml) and R848 (InvivoGen, San Diego, CA, 2.5 μg/ml).
7
Macrophage Immune Response to Herbal Extracts
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MDMs (in 200 µl of RPMI++) received filtered eluates of HC extracts (50 µl) at their non-cytotoxic concentration in duplicate settings. The cultures were incubated for 12, 24, and 36 h, then harvested and subjected to real-time PCR. Untreated and vehicle controls were MDMs receiving RPMI++ and vehicle solution, respectively. Positive control was MDMs stimulated with either polyinosinic:polycytidylic acid (poly IC, 5 μg/ml final; Sigma) or LPS from Escherichia coli O111:B4 (5 μg/ml final; Sigma). Poly IC was used as an inducer for gene expressions of myxovirus resistance 1 (Mx1), IRF3, IRF7, 2′-5′-oligoadenylatesynthetase 1 (OAS1), stimulator of interferon genes (STING), osteopontin (OPN), IFNα, IFNβ, IL-10, and TGFβ, while LPS was used as an inducer for gene expressions of IFNγ and TNFα (19 (link)).
8
Maternal Immune Activation Protocol
9
MAVS Overexpression and dsRNA Transfection
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Transient transfection of HeLa or 293-flp MAVS KD cells was performed as previously reported [20 (link)]. Briefly, these cells were seeded at a density of 1.5×105 or 3×105 cells per well, respectively, in 12-well culture plates for 16 to 20 h before transfection to reach 70 to 80% confluence.
To overexpress exogenous MAVS, TRAF2, and TRAF6, the cells were transfected with the MAVS expression vectors or an empty control vector using Lipofectamine LTX (Invitrogen). To introduce the foreign dsRNA polyinosinic-polycytidylic acid (poly I:C) (Sigma-Aldrich), the cells were transfected using Tranfectin (Bio-Rad). These cells were then incubated for the indicated period of time, depending on the experiment.
To overexpress exogenous MAVS, TRAF2, and TRAF6, the cells were transfected with the MAVS expression vectors or an empty control vector using Lipofectamine LTX (Invitrogen). To introduce the foreign dsRNA polyinosinic-polycytidylic acid (poly I:C) (Sigma-Aldrich), the cells were transfected using Tranfectin (Bio-Rad). These cells were then incubated for the indicated period of time, depending on the experiment.
10
Microglia Cell Line Characterization
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Although there are limitations of using any cell line, the utility of immortalized BV2 microglia as a suitable substitute for primary microglia has been shown (Henn et al., 2009 (link)). BV2 cells were cultured in DMEM (Invitrogen) supplemented with 5% fetal bovine serum and 1% penicillin/streptomycin at 37°C in a humidified incubator under 5% CO2. Cells were seeded at a density of 2.5 × 105 cells/ml 24 h before stimulations at which point cells were >90% confluent. Prior to stimulation, cells were washed twice with DPBS (Invitrogen) and cultured in serum-free DMEM for at least 1 h. Cells were stimulated for 24 h with one of the following: LPS (E. coli 0127:B8 1 μg/ml; Sigma-Aldrich); recombinant mouse IL-4 (20 ng/ml; R&D Systems); Pam3CSK4 (5 μg/ml; Invivogen); polyinosinic-polycytidylic acid (poly(I:C)) (50 μg/ml; Sigma-Aldrich); CpG oligodeoxynucleotides (CpG ODN) (1 μM; Enzo Life Sciences); or DPBS. In certain experiments, cells were pre-incubated with inhibitors (Merck Millipore) of IκB kinase (BMS-345541, 1 or 10 μM), PI3 kinase (LY294002, 10 μM), ERK1/2 kinase (MEK1/2) (PD98059, 10 μM), p38 MAP kinase (SB203580, 10 μM), c-Jun N-terminal kinase (SP600125, 20 μM) or equivalent volume of DMSO for 30 min prior to LPS stimulation.
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