Tlr agonists

Manufactured by InvivoGen

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TLR agonists are a class of immunomodulatory compounds that activate Toll-like receptors (TLRs). TLRs are a family of pattern recognition receptors that play a crucial role in the innate immune response. TLR agonists can stimulate specific TLR subtypes, leading to the activation of immune cells and the production of cytokines and other signaling molecules.

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Mouse TLR 1–9 agonist kit (5 citations) Human TLR agonist kit (3 citations)

15 protocols using tlr agonists

TLR Agonists Enhance Cytokine Secretion in AML

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The following TLR-agonists (InvivoGen; San Diego, CA, USA, if not stated otherwise) were used at following concentrations: (i) 10 ng/mL of the TLR1/2 heterodimer agonist Pam3CSK4, a synthetic triacetylated lipopeptide, (ii) 10 ng/mL of the TLR4 agonist LPS isolated from Escherichia coli 0111:B4 (Sigma Aldrich; St. Louis, MO, USA), (iii) 100 ng/mL of the TLR5 agonist flagellin isolated from Salmonella typhimurium, and (iv) 100 ng/mL of the dual TLR7/8 agonist resiquimod (R848). These compounds were tested on ten unselected AML patients at concentrations of 0.01, 0.1 and 1.0 µg/mL (Pam3CSK4, LPS and flagellin) or respectively of 0.1, 1.0 and 10 µg/mL (R848). At the selected concentrations, the agonists significantly increased the AML blasts’ secretion of the cytokines CCL2, CCL3, CCL4, CXCL8 and IL-6, while the proliferation capacity of the cells was not compromised (Figure S3).

Brenner A.K, & Bruserud Ø. (2019). Functional Toll-Like Receptors (TLRs) Are Expressed by a Majority of Primary Human Acute Myeloid Leukemia Cells and Inducibility of the TLR Signaling Pathway Is Associated with a More Favorable Phenotype. Cancers, 11(7), 973.

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OT-1 Mouse Splenocyte Activation and Cytokine Analysis

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Splenocytes were isolated from OT-1 mice after red blood cell osmotic lysis (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM EDTA). Splenocytes were cultured at 2×10⁶/mL in RPMI 1640 medium supplemented with L-glutamine, 10% fetal calf serum (FCS), 200 U/mL penicillin/streptomycin, 1% sodium-pyruvate, 1% HEPES, 50μM β-MeOH, and 2 µg/mL of the designated peptide. TLR agonists were purchased from InvivoGen (San Diego, CA) and added one hour before peptides at the following concentrations: 300 ng/ml Pam3CSK4, 10 µg/mL Poly(I:C) HMW, 10 μg/ml MPLAs, 3 µg/ml Gardiquimod, 10 µg/ml R848, 5 µM ODN 1826. At the time points indicated, cells were stained, fixed and frozen. Cells were thawed, washed and resuspended in PBS + 3% FCS + 1mM EDTA for flow cytometry. Cells were stained for 30 min at 4°C in a 1:4 dilution of brilliant stain buffer (BD 563794) in PBS + 3% FCS + 1mM EDTA. Intracellular cytokine staining was performed using standard procedures (8 (link)). In order to assess costimulation, blocking antibodies for CD80 (0.6 µg/mL), CD86 (0.25 µg/mL) or OX40L (0.6 µg/mL) were added 1 hour prior to antigen.

Zahm C.D., Colluru V.T., McIlwain S.J., Ong I.M, & McNeel D.G. (2018). TLR Stimulation During T-cell Activation Lowers PD-1 Expression on CD8+ T Cells. Cancer immunology research, 6(11), 1364-1374.

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CEP Modification of Albumin and Proteins

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CEP-adduction of proteins was prepared from commercially available highly purified cell-culture grade bovine serum albumin, human serum albumin, and transferrin (Sigma-Aldrich, St. Louis, MO). Proteins were CEP-adducted following previously published procedures [20] (link). Chromogenic limulus amebocyte lysate assay (Lonza, Walkersville, MD) was conducted to ensure protein preparations were endotoxin free (i.e., had completely undetectable endotoxin levels). Proper CEP adduction was confirmed using ELISA and western blot. All of the TLR-agonists, heat-killed bacteria, ATP, anti-TLR1 (rat polyclonal) antibody, and PMA were purchased from Invivogen (San Diego, CA). IL-4 was purchased from eBiosciences (San Diego, CA). MDA-BSA was kindly provided by Cristoph Binder (Medical University of Vienna). Hsp70 was purchased from Enzo Life Sciences (Farmingdale, NY) and HMGB1 was purchased from HMGBiotech (Milano, Itlay). The anti-CEP antibody used for the immuno-depletion experiments was produced by our lab in conjunction with Genscript (Piscataway, NJ) and was tested for specificity against a variety of CEP-modified proteins and lacked recognition to albumin and several other lipid-derived modifications.

Saeed A.M., Duffort S., Ivanov D., Wang H., Laird J.M., Salomon R.G., Cruz-Guilloty F, & Perez V.L. (2014). The Oxidative Stress Product Carboxyethylpyrrole Potentiates TLR2/TLR1 Inflammatory Signaling in Macrophages. PLoS ONE, 9(9), e106421.

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Intracellular TNF-α Detection in Monocytes

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Intracellular staining of monocytes for detection of TNF-α was performed after stimulation of 1 million/mL monocytes with EVs as previously described (7 (link)). To test the efficacy of anti-TLR neutralizing antibodies, one million PBMCs were treated with 1 µg/mL TLR1, TLR2, or TLR4 antibodies for 1 h, and then stimulated with TLR agonists (InvivoGen) for 16 h. Synthetic tripalmitoylated lipopeptide Pam3CysSerLys4 (Pam3CSK4, 20 ng/mL) was used as a TLR1 and TLR2 agonist, and LPS (5 ng/mL) was used as a TLR4 agonist. Finally, cells were stained with CD14-APC/Cy7 antibody (BioLegend) before permeabilization, then washed and stained for TNF-α, and run on the flow cytometer.

Danesh A., Inglis H.C., Abdel-Mohsen M., Deng X., Adelman A., Schechtman K.B., Heitman J.W., Vilardi R., Shah A., Keating S.M., Cohen M.J., Jacobs E.S., Pillai S.K., Lacroix J., Spinella P.C, & Norris P.J. (2018). Granulocyte-Derived Extracellular Vesicles Activate Monocytes and Are Associated With Mortality in Intensive Care Unit Patients. Frontiers in Immunology, 9, 956.

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Stimulation of HNECs with TLR Agonists

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HNECs were seeded onto 6-well dishes coated with collagen type I (Kurabo, Osaka, Japan) at 0.6 × 10⁶ cells/well for 24 hours prior to subsequent analysis. The cells were washed with phosphate-buffered saline (PBS), followed by the addition of TLR agonists (Invivogen, San Diego, CA, USA) for 24 hours, unless otherwise described. The following dosages were used: TLR1/2 (Pam3CSK4) 1 µg/ml, TLR2 (HKLM) 10⁸ cells/ml, TLR3 (Poly(I:C)) 10 µg/ml, TLR4 (LPS) 10 µg/ml, TLR5 (Flagellin) 10 µg/ml, TLR6/2 (FSL-1) 1 µg/ml, TLR7 (Imiquimod) 10 µg/ml, TLR8 (ssRNA40) 10 µg/ml, and TLR9 (ODN2006) 5 µM. In some experiments, fluticasone propionate (FP; Cayman Chemical Company, Ann Arbor, MI, USA) and JSH-23 (NFκB transcriptional activity inhibitor; Abcam, Cambridge, MA, USA), was added to the cells, 1 hour prior to TLR stimulation. The following dosages were used: FP 10 nM and JSH-23 30 µM, unless otherwise described. FP and JSH-23 were dissolved in dimethyl sulfoxide (DMSO). In experiments where FP or JSH-23 was used, corresponding DMSO control solutions were also used in parallel as controls.

Nakazono A., Nakamaru Y., Ramezanpour M., Kondo T., Watanabe M., Hatakeyama S., Kimura S., Honma A., Wormald P.J., Vreugde S., Suzuki M, & Homma A. (2021). Fluticasone Propionate Suppresses Poly(I:C)-Induced ACE2 in Primary Human Nasal Epithelial Cells. Frontiers in Cellular and Infection Microbiology, 11, 655666.

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Antibodies and Reagents for Immunological Assays

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Fluorochrome‐conjugated antibodies were from eBiosciences (Hatfield, UK), anti‐CD210 (IL‐10R) and control from Biolegend (London, UK) [LEAF purified (low endotoxin, azide‐free)] and rabbit anti‐streptavidin antibodies from Abcam (Cambridge, UK). iC3b, Syk inhibitor IV and Leukadherin‐1 were from Calbiochem (Beeston, UK). Salt solutions and media were from Lifetech (Paisley, UK) except lymphocyte growth medium from Clonetics (Wokingham, UK). Polystyrene microspheres were from Spherotech (Sheffield, UK), IL‐12 and IL‐15 from Peprotech (London, UK), IL‐18 from R&D Systems (Abingdon, UK) and TLR agonists from Invivogen (San Diego, California, USA). Cytokines were quantified by cytometric bead array from BD Biosciences (San Jose, California, USA). Phosflow reagents were from BD Biosciences.

Roberts A.L., Fürnrohr B.G., Vyse T.J, & Rhodes B. (2016). The complement receptor 3 (CD11b/CD18) agonist Leukadherin‐1 suppresses human innate inflammatory signalling. Clinical and Experimental Immunology, 185(3), 361-371.

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Modulation of Alveolar Macrophage Responses

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The effect of SCGB1a1 protein on AM responsiveness to inflammatory stimuli were studied in vitro. Flow sorted AMs were plated (50 (link)) in 12-well plates at 1 × 10⁵ cells/well in triplicates and were incubated with Toll-like receptor (TLR) agonists (Invivogen) 2- heat-killed Listeria monocytogenes (HKLM), TLR4-Lipopolysaccharide from Escherichia coli K12 (LPS) and TLR5- Salmonella typhimurium Flagellin (FLA) in presence or absence of recombinant SCGB1a1 protein at 5 μg/mL (Creative BioMart). Culture supernatant was collected and total RNA was isolated at 72 h post-stimulation. The release of cytokines and chemokines in culture supernatant was analyzed by a Bio-Plex 200 system (Bio-Rad) using multiplex immunoassays.

Xu M., Yang W., Wang X, & Nayak D.K. (2020). Lung Secretoglobin Scgb1a1 Influences Alveolar Macrophage-Mediated Inflammation and Immunity. Frontiers in Immunology, 11, 584310.

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Preparation of Complete Cell Culture Media

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Complete culture media (CM) contained RPMI 1640, 1% L-glutamine, 1% penicillin/streptomycin, 50 μM 2β-Mercaptoethanol, 1% sodium-pyruvate, 1% non-essential amino acids and heat-inactivated 10% Fetal Calf Serum (FCS). Recombinant human Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF), Interleukin (IL)-4, IL-12 and IL-2 were purchased from Peprotech (Rocky Hill, NJ). TLR agonists were purchased from Invivogen (San Diego, CA). Media for staining cells contained PBS 1X without Ca²⁺/Mg²⁺, heat-inactivated 2% FCS and 2 mM EDTA.

Nouri-Shirazi M., Tamjidi S., Nourishirazi E, & Guinet E. (2017). TLR8 combined withTLR3 or TLR4 agonists enhances DC-NK driven effector Tc1 cells. Immunology letters, 193, 58-66.

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Isolation and Differentiation of Human Keratinocytes

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Human primary KCs were isolated as described previously [47 (link)] and cultured in serum-free KC growth medium (KGM-2, Lonza, Basel, Switzerland). Primary human KCs from three different adult donors were used for preparation of organotypic skin cultures as described previously [48 (link)] and, in parallel, cultured in two-dimensional monolayers. For analyzing proliferating KCs, 2nd passage cells at 50% confluency were used. Differentiation was induced by maintaining confluent KCs for 7 days at high calcium (1.1 mM Ca²⁺) conditions. The quality of keratinocyte differentiation was validated by analysis of well-known differentiation markers, including keratin 1, keratin 10, the S100 genes S100A7 and A9, desmoglein-1 and keratinocyte differentiation-associated protein. For in vitro assays, cytokines in the following final concentrations were used: IL-1β (10 ng/mL), TNF-α (10 ng/mL), IL-17 (10 ng/mL), INF-γ (100 ng/mL) (all R&D System, Minneapolis, MN, USA) and toll-like receptor (TLR) agonists (InvivoGen, Toulouse, France).

Beer L., Kalinina P., Köcher M., Laggner M., Jeitler M., Abbas Zadeh S., Copic D., Tschachler E, & Mildner M. (2020). miR-155 Contributes to Normal Keratinocyte Differentiation and Is Upregulated in the Epidermis of Psoriatic Skin Lesions. International Journal of Molecular Sciences, 21(23), 9288.

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TLR Agonist Treatment of THP-1 Cells

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THP-1 cells were treated with the following TLR agonists from InvivoGen for 2 h (n=3 per group) at the concentrations listed; PAM3CSK4 (100 ng ml⁻¹), HKLM (10⁸ cells ml⁻¹), Poly(I:C) (10 mg ml⁻¹), Poly(I:C) LMW (10 mg ml⁻¹), Flagellin (100 ng ml⁻¹), FSL-1 (100 ng ml⁻¹), Imiquimod (5 μg ml⁻¹), ssRNA40 (1 μg ml⁻¹) and ODN2006 (2 μM). THP-1 cells were also treated with buffer as a control, LPS (1 μg ml⁻¹, E. coli 055:B5, Sigma-Aldrich) and IL-1β (10 ng ml⁻¹, recombinant, E. coli, Sigma-Aldrich).

IIott N.E., Heward J.A., Roux B., Tsitsiou E., Fenwick P.S., Lenzi L., Goodhead I., Hertz-Fowler C., Heger A., Hall N., Donnelly L.E., Sims D, & Lindsay M.A. (2014). Long non-coding RNAs and enhancer RNAs regulate the lipopolysaccharide-induced inflammatory response in human monocytes. Nature Communications, 5, 3979.

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