Ifnb−/− mice and Ifnar1−/− mice were gifted from Genhong Cheng Laboratory (University of California, CA, USA). Tlr4−/− mice were purchased from Model Animal Research Center (Nanjing, China). Wild-type (WT) C57BL/6 mice were acquired from Vital River Laboratory Animal Technology (Beijing, China). All the mice were maintained in the specific pathogen-free (SPF) environment at Suzhou Institute of Systems Medicine (ISM) under a controlled temperature (25°C) and a 12-h day/night cycle. Male 8–10-week-old mice were used in all the experimental AP mice models. All mice experiments were undertaken in accordance with the US National Institutes of Health Guide for the Care and Use of Laboratory Animals, with the approval of the Scientific Investigation Board of ISM, Suzhou. Antibodies against HO-1 (#70081) and GAPDH (#5174) were from Cell Signaling Technology (Danvers, MA). NQO1 antibody (#ab2346) and KEAP1 antibody (#ab119403) were from Abcam (Cambridge, MA). Caerulein and IFNAR inhibitor were from MCE (Monmouth Junction, NJ). L-Arginine was from Sigma-Aldrich (St. Louis, MO). PolyI:C was from Thermo Fisher Scientific (Waltham, MA). Recombinant mouse IFN-β was from R&D Systems (Minneapolis, MN). Taurocholate was from SolarBio (Beijing, China).
Polyi c
Manufactured by Thermo Fisher Scientific
Sourced in United States
PolyI:C is a synthetic double-stranded RNA molecule that mimics the structure of viral RNA. It is commonly used as an immunostimulant in cell culture and animal research applications.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Recombinant mouse ifn β, by R&D Systems (1 mentions)
Wild type c57bl 6 mice, by Charles River Laboratories (1 mentions)
Taurocholate, by Solarbio (1 mentions)
L arginine, by Merck Group (1 mentions)
Phosphorimager screen, by GE Healthcare (1 mentions)
Nextseq 500 system, by Illumina (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Poly 1 c, by Bio-Techne (1 mentions)
Poly da dt, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Thermo Fisher Scientific (1 mentions)
Poly da dt, by InvivoGen (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Ifn β, by R&D Systems (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Beas 2b, by American Type Culture Collection (1 mentions)
Nci h460, by American Type Culture Collection (1 mentions)
Cpg1826, by Merck Group (1 mentions)
Lipopolysaccharide lps, by Merck Group (1 mentions)
9 protocols using polyi c
1
Acute Pancreatitis Murine Model
2
Quantifying Protein-RNA Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Five micrograms of protein lysate was incubated with 1.4 pmol of radiolabeled probe in a final volume of 20 μl. Reactions were incubated at 22°C for 20 min in 1× reaction buffer consisting of 100 mM KCl, 10 mM Tris pH 7.6, 5 mM MgCl2, 1 mM DTT, 500 ng Poly (I)-(C) (ThermoFisher Waltham, MA, USA). Reactions with unlabeled specific wild-type (wt) competitor and non-specific tRNA competitor at 10, 25, 50 and 100× molar excess were carried out in parallel. The 20 μl reaction was loaded onto a 1× Tris Borate EDTA buffer, 6% non-denaturing polyacrylamide gel and electrophoresed for 2.5 h. Gels were transferred to Whatman 3CHR paper (Whatman, Maidstone UK), dried at 80°C under vacuum for 2 h, and exposed to a phosphorimager screen (GE Healthcare Life Sciences, Little Chalfont, UK). At least three independent assays were performed per probe. Typhoon 9410 software was used to analyze the gels and determine relative signal intensities of the shifted bands. Background subtracted signal intensity for each sample was determined as a ratio to wt in the absence of competitor. Relative average intensities were calculated and compared by paired t-tests using GraphPad Prism.
3
Transcriptomic Analysis of innate immune responses
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ncgRNA and PAF1 KO cells were plated at a density of 6.0 × 105 cells per well in 6-well plates and incubated at 37 °C for 24 hours. Cells were then stimulated with poly I:C (2.0 μg/mL, Tocris), poly dA:dT (1.0 μg/mL, InvivoGen), LPS (1.0 μg/mL, eBioscience), IFN-β (50 U/mL, R&D Systems), and TNF-α (100 ng/mL, Gibco Thermofisher) at the given concentrations. For poly I:C and poly dA:dT, transfection was performed with Lipofectamine 2000 (ThermoFisher) per manufacturer instruction. After 3 hours post-stimulation, all samples were collected with Trizol reagent (Ambion) and stored at − 80 °C. Total cellular RNA was extracted and purified with the RNeasy mini kit (Qiagen). At the sequencing facility, RNA samples were prepared with the QuantSeq3’ mRNA-Seq Library kit for Illumina (Lexogen GmbH), following a standard protocol [84 (link)]. The resulting cDNA libraries were sequenced on an Illumina NextSeq500 system with approximately 4 million reads per sample.
4
TLR3 Agonist Treatment of Lung Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The lung cancer cell lines (A549 and NCI-H460) and human normal bronchial epithelial cell BEAS-2B were all bought from American Type Culture Collection (ATCC). A549 and NCI-H460 cells were cultured in RPMI-1640 medium and BEAS-2B cells was cultured in DMEM medium. All mediums were mixed with 10% fetal bovine serum (FBS; Life Technologies, NY, USA) and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) in a humidified incubator with 5% CO2 at 37°C. For TLR3 agonist treatment, 10 and 50 µg/ml Poly (I:C) (ThermoFisher, USA) were used to treat A549 cells for 48 h.
5
Whole Tumor Cell Vaccination Induces Immunity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole tumor cell vaccination experiments were conducted via s.c. injection of 10 × 106 50 Gy-irradiated SCC VII cells in 1× HBSS with 50 µg polyI:C (Thermo Fisher). Immunized mice were challenged 14 d later by s.c. injection of 5 × 105 live SCC VII cells.
6
Agonist Preparation for Cell Stimulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Poly I:C was obtained from Thermo Fisher Scientific (Milwaukee, WS). Pam3CSK4 was obtained from EMC Microcollections (Tuebingen, Germany). Lipopolysaccharide (LPS) and CpG 1826 were purchased from Sigma-Aldrich (St. Louis, MO). All agonists were reconstituted in water at the concentration recommended by manufacturer; for cell stimulation, appropriate dilutions were prepared in complete medium.
7
Formulation of Vaccine Adjuvant Combination
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Vaccine antigens, colanic acid (CA), CA-tgD and CA-CRM197 were formulated with triple combination adjuvant consisting of Polyinosinic:polycytidylic acid (Poly (I:C)) (Thermo Fisher Scientific), host defense peptide (HDP) and polyphosphazene (PCEP) (Idaho National laboratory) immediately prior to administration. Formulations were prepared by first mixing 10 µg of PIC and 20 µg HDP in PBS and incubating at room temperature (RT) for 15 min. CA, CA-tgD or CA-CRM197 were added before the addition of 10 µg of PCEP, making a final ratio of 1:2:1 of PIC:HDP:PCEP (TriAdj). Mixtures were incubated in the dark for 15 min at RT prior to administration.
8
Inducing Stress Granules with PolyI:C and Arsenite
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PolyI:C (Sigma, St. Louis, MO, USA) was dissolved in RNase-free water containing 0.98% NaCl to make 5 mg/mL of stock solution. Before use, PolyI:C was incubated at 50 °C for 20 min followed by slow cooling to room temperature for annealing. To mimic stress induced by the viral replication of intermediate dsRNA, 1 μg/mL of PolyI:C was transfected into the cells with lipofectamine 2000 (ThermoFisher, Waltham, MA, USA) for 9 h or the indicated time. To induce oxidative stress, the cells were treated with 0.5 mM AS (Sigma, St. Louis, MO, USA) for 45 min. G3BP1 was used as a marker of PolyI:C or AS-induced SGs. For SG counting, the percentage of cells with SGs was analyzed in 50 cells per condition and a minimum of three granules per cell were required to score as positive.
9
Macrophage Polarization Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CA-Mφ were generated by priming BMM with 150 U/ml IFN-γ (R&D Systems, Minneapolis, MN) for 6 h, and then stimulating cells overnight with 150 U/ml IFN-γ and 100 ng/ml LPS (from Salmonella typhimurium; no. 225; List Biological Laboratories, Campbell, CA). AA-Mφ were generated by stimulating macrophages overnight with 20 ng/ml IL-4 (R&D Systems, Minneapolis, MN). Reg-Mφ were generated by stimulating macrophages overnight with 100 ng/ml LPS and either 200 nM PGE2 (Cayman Chemical, Ann Arbor, MI) or 200 μM adenosine (Sigma-Aldrich, St. Louis, MO). Studies of macrophages stimulated overnight with TLR agonists were performed with the following reagents: Pam3CSK4 (100 ng/ml); poly(I:C) (10 µg/ml); ultrapure flagellin from Salmonella typhimurium (FLA-ST; 100 ng/ml); R848 (100 ng/ml), ODN 1826 (1 µM). All TLR agonists were purchased from Invivogen (San Diego, CA) except for poly(I:C), which was purchased from Tocris (Bristol, United Kingdom).
For experiments in which both RNA isolation and cytokine analyses were performed, 6 x 10 6 cells were plated onto 60-mm dishes (ThermoFisher, Waltham, MA). For experiments in which cytokine analyses were performed and RNA was not isolated, 1 x 10 5 cells were plated onto 24-well plates (ThermoFisher). For assays of lysosomal damage, 8 x 10 4 cells were plated onto 35-mm dishes with attached 14-mm coverglass (MatTek Corporation, Ashland, MA).
For experiments in which both RNA isolation and cytokine analyses were performed, 6 x 10 6 cells were plated onto 60-mm dishes (ThermoFisher, Waltham, MA). For experiments in which cytokine analyses were performed and RNA was not isolated, 1 x 10 5 cells were plated onto 24-well plates (ThermoFisher). For assays of lysosomal damage, 8 x 10 4 cells were plated onto 35-mm dishes with attached 14-mm coverglass (MatTek Corporation, Ashland, MA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!