Poly (I:C) (TLR3 ligand) was purchased from InVivogen (San Diego, CA, USA) and dissolved in endotoxin and nuclease free water and kept at −20°C until use. DF-1 cells were treated at the concentrations of 0.1, 1, 5, and 10 μg/mL for 24 h. Also, to determine the time kinetics of mRNA expression, cells were treated for 1, 3, 6, 12, and 24 h at 10 μg/mL concentration. BAY 11–7085 and Tanshione IIA were purchased from Sigma-Aldrich (Louis, MO, USA). BAY 11-7085 (BAY) inhibits the IκBα phosphorylation, resulting in prevention of NFκB activation, was used as NFκB inhibitor. Tanshinone IIA (Tan-II) may inhibit the binding of AP-1 to DNA in chicken heteophils [19 (link)]. DF-1 cells were pretreated with BAY 11–7085 (5 μM) and Tan-II (25 μM), for 3 h, of which concentrations and time were determined from the preliminary experiment in this study, and then stimulated with poly (I:C) (5 μg/mL) for 3 h.
Tlr3 ligand poly 1 c
Manufactured by InvivoGen
Sourced in United States
TLR3 ligand poly (I:C) is a laboratory product designed to activate the Toll-like receptor 3 (TLR3) signaling pathway. It is a synthetic double-stranded RNA (dsRNA) molecule that mimics the structure of viral RNA, triggering an immune response. The core function of this product is to serve as a tool for research purposes, facilitating the study of TLR3-mediated signaling and its implications in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi medium, by Thermo Fisher Scientific (2 mentions)
Bay 11 7085, by Merck Group (1 mentions)
Venetoclax, by Selleck Chemicals (1 mentions)
Penicillin streptomycin, by Corning (1 mentions)
L glutamine, by Corning (1 mentions)
Il 15, by Miltenyi Biotec (1 mentions)
Hepes buffer, by Corning (1 mentions)
Human il 17a, by R&D Systems (1 mentions)
Recombinant murine gm csf, by R&D Systems (1 mentions)
Murine il 4, by R&D Systems (1 mentions)
Phospho nf κb, by Cell Signaling Technology (1 mentions)
Jnk1 2, by Cell Signaling Technology (1 mentions)
Phospho creb, by Cell Signaling Technology (1 mentions)
Phospho jnk1 2, by Cell Signaling Technology (1 mentions)
Cpg odn, by InvivoGen (1 mentions)
Phospho p38, by Cell Signaling Technology (1 mentions)
U0126, by InvivoGen (1 mentions)
Phospho erk1 2, by Cell Signaling Technology (1 mentions)
Phospho akt ser473, by Cell Signaling Technology (1 mentions)
Nf κb, by Cell Signaling Technology (1 mentions)
8 protocols using tlr3 ligand poly 1 c
1
TLR3-Mediated Inflammatory Response in Chicken Cells
2
Heat-Inactivated Viral Antigen Stimulation
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Heat-inactivated Influenza B Brisbane (7,4*10x3 K/mL TCID50), heat-inactivated Influenza H1N1 California (3,24 x 10x5 K/mL TCID50), and heat-inactivated SARS-CoV-2 Wuhan-Hu-1 variant (1,4 x 10x3K/mL TCID50) were used in the study. Viruses had been heat-inactivated for 30 minutes at 60°C. Heat-inactivation had been checked for sufficiency by cell culture inoculation and subsequent qPCR testing. R848 (Resiquimod, TLR7/8 ligand) (Invivogen, San Diego, CA, USA) (3 μg/mL); Poly I:C (TLR3 ligand) (Invivogen, San Diego, CA, USA) (10 μg/mL).
3
Inactivated H9N2 AIVs Vaccine Preparation
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H9N2 AIVs (A/Chicken/Jiangsu/DHN12/2013 (H9N2, HA clade: 4.2.5)) was obtained from the Guangdong Enterprise Key Laboratory of Biotechnology R&D of Veterinary Biologics. Briefly, the H9N2 vaccine candidate virus (50% egg infective dose, EID50 at 108/0.1 mL) was propagated in 10-day-old embryonated SPF chicken eggs (Guangdong Enterprise Key Laboratory of Biotechnology R&D of Veterinary Biologics). Virus titers were measured following the method of Reed & Muench as described previously [6 (link),21 (link)]. Virulent H9N2 AIVs was inactivated with 0.05% formaldehyde (Ferak Berlin Gmbh, Berlin, Germany) by incubating at 4°C for 12 h. Complete inactivation of the virus was confirmed through repeated passages in 10-day-old embryonated SPF chicken eggs and Madin-Darby canine kidney cells as previously described [6 (link),21 (link)]. The inactivated H9N2 AIVs antigen (H9N2 IAIV) was purified on a discontinuous sucrose density gradient as described [22 (link)].
The 1-day-old white Muscovy (Wens Foodstuff Group, China) was raised in an isolator to allow the animals to eat and drink normally. These ducks were raised for two weeks to adapt to their living environment. All duck serum samples were confirmed to be free of any AIV infection in the study. Adjuvant of TLR2 ligand pam2CSK4, TLR3 ligand poly (I:C) and TLR7 ligand imiquimod were purchased from InvivoGen (San Diego, CA).
The 1-day-old white Muscovy (Wens Foodstuff Group, China) was raised in an isolator to allow the animals to eat and drink normally. These ducks were raised for two weeks to adapt to their living environment. All duck serum samples were confirmed to be free of any AIV infection in the study. Adjuvant of TLR2 ligand pam2CSK4, TLR3 ligand poly (I:C) and TLR7 ligand imiquimod were purchased from InvivoGen (San Diego, CA).
4
PBMC and DC Activation Assay
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Isolated NK cells or total PBMCs were cultured in medium only (negative control) or with 10 ng/mL IL-15 (Miltenyi) for 24 hours. When indicated, BCL2 inhibitor Venetoclax (Selleckchem) was added at a concentration of 100nM. Isolated mDCs were cultured in medium only (negative control) or with 2 μg/mL TLR3 ligand Poly(I:C) (Invivogen) for 24 hours. K562 were cultured in medium only for expansion. The cells were cultured in RPMI medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (MilliporeSigma), 1% l-glutamine (Corning), 1% penicillin/streptomycin (Corning), and 1% HEPES buffer (Corning) and kept at 37°C in 5% CO2 during the culture.
5
Recombinant Cytokine Stimulation Assay
6
Signaling Pathways Activated by TLR Ligands
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TLR2 ligand Pam3CSK4, TLR3 ligand Poly(I:C), TLR4 ligand LPS, TLR7/8 ligand CL087, TLR9 ligand CpG ODN, p38 inhibitor SB2021980, and ERK1/2 inhibitor U0126 were purchased from InvivoGen (San Diego, CA, USA). High-mobility group box 1 protein (HMGB1) was from Sigma-Aldrich (St. Louis, MO, USA). Kinases inhibitors including SB203580, PD98059, SP600125, and LY294002 were purchased from Calbiochem (San Diego, CA, USA); and antibodies against p38, phospho-p38, ERK1/2, phospho-ERK1/2, Akt, phospho-Akt (Ser473), JNK1/2, phospho-JNK1/2, CREB, phospho-CREB, NF-κB, and phospho-NF-κB were obtained from Cell Signaling (Danvers, MA, USA).
7
Lipid-mediated Nucleic Acid Delivery Protocols
8
Profiling lncRNA Expression in Immune Cells
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The human monocytic cell lines, THP-1 cells (ATCC TIB-202; American Type Culture Collection, Rockville, Md.), were grown in lipopolysaccharide (LPS)-free RPMI medium (Gibco, ThermoFisher Scientific, USA) supplemented with 10% fetal bovine serum in an incubator containing 5% CO 2 at 37°C. One million cells cultured in LPS-free RPMI medium were treated with TLR3 ligand poly(I:C) (Invivo-Gen, California, USA) or TLR4 ligand LPS (Sigma-Aldrich, Merk, Darmstat, Germany) for 4 hours and 24 hours respectively. PBMCs derived from healthy controls were rested overnight in RPMI medium at 37°C incubator, and were separated into the adherent monocytes and the suspended lymphocytes. Poly(I:C) and LPS stimulation experiments were also performed on primary monocytes and lymphocytes. RNAs were then extracted from THP-1 cells, primary human monocytes and lymphocytes for further qRT-PCR analyses. The difference in expression of the target gene relative to averaged internal control gene was calculated using the comparative threshold cycle (Ct) method and evaluated by ΔCT (CT lncRNA -CT GAPDH ). The fold of expression of lncRNAs was calculated by 2^-ΔΔCT [(CT lncRNA -CT GAPDH ) -median ΔCT of control replicates].
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