Resiquimod r848

Manufactured by InvivoGen

Sourced in United States, France

Resiquimod (R848) is a synthetic small-molecule agonist for Toll-like receptor 7 (TLR7) and TLR8. It functions as an immunomodulatory agent that can activate the innate immune response.

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Lab products found in correlation

Lipopolysaccharide lps, by InvivoGen (4 mentions) Poly 1 c, by InvivoGen (3 mentions) Cpg a, by InvivoGen (3 mentions) Polyinosinic polycytidylic acid, by InvivoGen (3 mentions) Cgamp, by InvivoGen (3 mentions) Rpmi 1640, by Thermo Fisher Scientific (3 mentions) Dimethyl sulfoxide (dmso), by Merck Group (3 mentions) Escherichia coli lipopolysaccharide o26 b6, by Merck Group (2 mentions) Class b cpg odn 1826, by InvivoGen (2 mentions) Cd43 microbead, by Miltenyi Biotec (2 mentions) Pam3csk4, by InvivoGen (2 mentions) Muramyl dipeptide mdp, by InvivoGen (2 mentions) Lipopolysaccharides (lps), by Merck Group (2 mentions) Fetal bovine serum (fbs), by Biowest (2 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) C57bl 6j female mice, by Jackson ImmunoResearch (1 mentions) Imject alum, by Thermo Fisher Scientific (1 mentions) Quil a, by InvivoGen (1 mentions) Penicillin, by Thermo Fisher Scientific (1 mentions) Streptomycin, by Thermo Fisher Scientific (1 mentions)

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Resiquimod (45 citations)

37 protocols using resiquimod r848

Murine PDAC Cell Lines and Treatments

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The murine PDAC KCKO and luciferase-expressing KCKO (KCKO-Luc) cell lines were a gift from Dr Pinku Mukherjee (University of North Carolina, Charlotte, North Carolina, USA, 2010). OVA-expressing KCKO (KCKO-OVA) and KP2 cell lines were generously provided by Dr David Denardo (Washington University of Medicine, St. Louis, Missouri, USA, 2016). Luciferase-expressing KP2 cells (KP2.1-Luc) were generated by transfecting the KP2 cells with luciferase-containing vectors. All cell lines were negative for Mycoplasma and cultured in RPMI1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. All the cell lines used for experiments were within three passages of subsequent culture.
R848 (resiquimod) was purchased from Invivogen (cat# tlrl-r848-5). For in vitro experiments, 5 µg/mL of R848 were added in the complete culture medium. For in vivo experiments, 3 mg/kg were administered by retro-orbital injections (diluted in PBS, 100 µL per mouse) 1 day before, 1 day after, and 1 week after SBRT treatments (total of 3 times). For CD8⁺ T cell depletion, 200 µg of anti-CD8 (clone: 53–6.7) antibody or isotype rat immunoglobulin G (IgG) (diluted in phosphate buffered saline (PBS), 100 µL per mouse) were administered intraperitoneally every 3 days.

Ye J., Mills B.N., Qin S.S., Garrett-Larsen J., Murphy J.D., Uccello T.P., Han B.J., Vrooman T.G., Johnston C.J., Lord E.M., Belt B.A., Linehan D.C, & Gerber S.A. (2022). Toll-like receptor 7/8 agonist R848 alters the immune tumor microenvironment and enhances SBRT-induced antitumor efficacy in murine models of pancreatic cancer. Journal for Immunotherapy of Cancer, 10(7), e004784.

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Chikungunya VLP Vaccine Immunization

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Female C57BL/6J mice were obtained from the Jackson Laboratory [Bar Harbor, ME, USA] at 6–8 weeks of age for studies in adult mice. Female C57BL/6J mice were also obtained at 12 months and allowed to age to at least 18 months for studies in aging mice. All procedures in the document were approved by the UGA Institutional Animal Care and Use Committee, # A2015 06-004-Y3-A12. Mice were immunized on days 0, 21, and 42 and blood samples were taken on days 0, 14, and 35 via the submandibular method using 5mm lancets.[18 (link)] Vaccines were formulated to contain 30μg [~0.3–0.4μg E2 content] chikungunya VLPs adjuvanted with 20μg QuilA [InvivoGen, San Diego, CA, USA], 10μg R848/Resiquimod [InvivoGen], 1:1 by volume Imject Alum [Thermo Fisher Scientific], or in PBS alone (no adjuvant). Vaccines were delivered via intramuscular injection to the hindlimb quadriceps in a total volume of 50μl or subcutaneous injection to the scruff of the neck in a total volume of 100μl.

Arévalo M.T., Huang Y., Jones C.A, & Ross T.M. (2019). Vaccination with a chikungunya virus-like particle vaccine exacerbates disease in aged mice. PLoS Neglected Tropical Diseases, 13(4), e0007316.

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Differentiation Assay for B-cell IgG Secretion

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To investigate the IgG secretion of total B cells, CD11c⁺ B cells, and CD11c^- B cells from the PB of GD patients, we established a controlled culture system to induce B cells to differentiate into ASCs. B cells, CD11c⁺ B cells, and CD11c^- B cells isolated from the PB of GD patients were cultured separately at a density of 1×10⁵ cells per well in a total volume of 200 μl in 96-well round-bottom plates. A TLR7 agonist (1 µg/ml, R848, resiquimod, In vivoGen, USA) and 100 U/ml IL-2 (PeproTech, USA) were added to costimulate the cells to drive the extrafollicular B-cell response for 9-12 days (27 (link)). For continuous detection of total IgG in the culture supernatant, the differentiation culture was extended up to 12 days, and the culture supernatant was collected for subsequent detection. The percentage of plasmablasts was detected by flow cytometry according to the procedure described above. The culture medium of the B-cell differentiation culture experiment was RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin, 2 mM L-glutamine (all from Gibco, USA), and Insulin Transferrin Selenium (ITS; 1.0 mg/ml insulin, 1.0 mg/ml transferrin, 3.4 µM selenium, ScienCell, USA). Experiments were performed at least three times with different donors.

Cao Y., Zhao X., You R., Zhang Y., Qu C., Huang Y., Yu Y., Gong Y., Cong T., Zhao E., Zhang L., Gao Y, & Zhang J. (2022). CD11c+ B Cells Participate in the Pathogenesis of Graves’ Disease by Secreting Thyroid Autoantibodies and Cytokines. Frontiers in Immunology, 13, 836347.

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HEK-Blue hTLR-8 Reporter Cell Assay

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HEK-Blue^TM hTLR-8 reporter cell lines and reagents, such as selection media Quanti-Blue^TM reagent, and the agonists single-stranded GU-rich oligonucleotide complexed with LyoVec (ssRNA40/LyoVec^TM) and R848 (resiquimod), were purchased from InvivoGen (Toulouse, France). This cell line carries a construct for SEAP coupled to the NF-κB/AP-1 promoter. The HEK-Blue^TM-hTLR8 cell line was maintained in DMEM (Life Technologies Europe B.V) containing 10% heat inactivated fetal bovine serum (FBS), L-glutamine (2.0 mM-Sigma-Aldrich Chemie B.V), glucose (4.5g/L Sigma-Aldrich Chemie B.V), penicillin-streptomycin (50 U/mL–50 μg/mL Sigma-Aldrich Chemie B.V), and normocyn (100 μg/mL Sigma-Aldrich Chemie B.V). HEK-Blue^TM hTLR8 cells were grown to approximately 80% of confluence. After culturing for 3 passages, all reporter cell lines were maintained on selection media according to the manufacturer’s protocol.

Figueroa-Lozano S., Valk-Weeber R.L., Akkerman R., Abdulahad W., van Leeuwen S.S., Dijkhuizen L, & de Vos P. (2020). Inhibitory Effects of Dietary N-Glycans From Bovine Lactoferrin on Toll-Like Receptor 8; Comparing Efficacy With Chloroquine. Frontiers in Immunology, 11, 790.

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CD40 Agonist and Adjuvant Effects on Serum Antibodies

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Serum was collected from young C57BL/6 mice prior to treatment to obtain baseline antibody levels. Mice were then treated with PBS and 50 μg rat IgG2a isotype control (Clone 2A3; BioXcell), or 50 μg rat anti-mouse CD40 (Clone FGK45; made in house) with either 100 μg of Poly(I:C) (Invivogen), 50 μg of Escherichia coli lipopolysaccharide O26:B6 (Sigma-Aldrich), 100 μg R848 (Resiquimod) (Invivogen), or 100 μg Class B CpG ODN 1826 (Invivogen). Serum was collected 7 days after treatment. Baseline and day 7 serum was then analyzed by ELISA as described in previous sections.

Agazio A., Cimons J., Shotts K.M., Guo K., Santiago M.L., Pelanda R, & Torres R.M. (2020). Histone H2A-Reactive B Cells Are Functionally Anergic in Healthy Mice With Potential to Provide Humoral Protection Against HIV-1. Frontiers in Immunology, 11, 1565.

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Stimulation of Murine B Cell IgM Production

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Untouched B cells were purified from C57BL/6 spleens using negative selection with CD43 MicroBeads (MACS Miltenyi Biotec). Purified B cells were added 2 × 10⁶ cells/ml to 24-well plates with a final volume of 1 ml. Cells were cultured in complete DMEM + 10% FBS with 20 ng/ml recombinant mouse BAFF (R&D Systems) in media alone or treated with 10 μg/ml of either Poly(I:C) (Invivogen), Escherichia coli lipopolysaccharide O26:B6 (Sigma-Aldrich), R848 (Resiquimod; Invivogen), or Class B CpG ODN 1826 (Invivogen). Culture supernatants were collected after incubation for 10 days and analyzed by ELISA for total IgM concentration, H2A-reactive IgM, Gp140-reactive IgM, and Chromatin-reactive IgM. For ELISAs with in vitro samples, plates were coated with 5 μg/ml of H2A or gp140, or 10 μg/ml of chromatin in PBS. Undiluted culture supernatants were added to each well, and ELISAs were completed as previously described. Data for antigen-reactive IgM is represented as 405 nm optical density values normalized to total IgM 405 nm optical density.

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PBMC Stimulation and DC Activation

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Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll gradient centrifugation (GE Healthcare, Little Chalfont, UK) from whole blood. PBMCs were cultured at a density of 1 × 10⁶ cells/mL in 24-well plates (Corning, New York City, NY, USA) in RPMI 1640 with 10% of Fetal Bovine Serum (FBS), in the presence or absence of Resiquimod (R848) (Invivogen, San Diego, CA, USA) (100 ng/mL) for 6 hours at 37 °C. Then, cells were collected and stained with specific antibodies discriminating viable pDCs and mDCs and evaluating their expression of activation markers. After the isolation, the cells were stained with specific antibodies enabling to discriminate mDCs and pDCs.

Corsetti M., Ruocco G., Ruggieri S., Gasperini C., Battistini L, & Volpe E. (2019). Resiquimod-Mediated Activation of Plasmacytoid Dendritic Cells Is Amplified in Multiple Sclerosis. International Journal of Molecular Sciences, 20(11), 2811.

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Bovine Immune Response to Pathogen Mimics

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The selected PAMPs were chosen to represent major disease-relevant pathogens in cattle. Two serotypes of lipopolysaccharide (LPS) extracted from Gram-negative bacteria were used (E. coli O55:B5 and E. coli O11:B4, Sigma Aldrich), as potent activator of TLR4. LPS strains were tested at concentrations between 0.1 and 2 µg/mL, based on previous reports^{35 (link),40 (link)}. Pam3CSK4 (Pam3CysSerLys4) is a synthetic triacylated lipopeptide, chosen to mimic the response of lipopeptide from Gram-positive bacteria, and which is a potent activator of TLR1/2. Pam3CSK4 (InvivoGen) was tested at concentrations between 0.5 and 10 µg/mL, chosen based on previous reports^{41 (link),42 (link)}. Imidazoquinoline, also known as resiquimod (R848) (InvivoGen), was chosen to mimic the immune response to single stranded viral RNA (InvivoGen), and is a potent activator of TLR7/8. R848 was tested at concentrations between 0.1 and 2 µg/mL, chosen based on previous reports^{43 (link)}.

Reid C., Beynon C., Kennedy E., O’Farrelly C, & Meade K.G. (2021). Bovine innate immune phenotyping via a standardized whole blood stimulation assay. Scientific Reports, 11, 17227.

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Isolation and stimulation of human monocytes

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Whole venous blood was collected into tubes containing 1.8 mg/ml K₂EDTA (Becton Dickinson, Oxford, UK). PBMCs were isolated using Ficoll-plaque gradients (Cedarlane, Burlington, ON, Canada) as previously described [17 (link)]. Monocytes were isolated from PBMCs using CD14⁺ selection beads (Miltenyi Biotec, Bisley, UK) as per the manufacturer’s instructions before being cultured in RPMI1640 medium supplemented with 5% (v/v) fetal calf serum (PAN-Biotec, Aidenbach, Germany) and 1% (v/v) penicillin–streptomycin solution (PAA, Pasching, Austria). Cells were incubated with or without 100 ng/ml PAM₃CSK₄ (PAM3) or 10 ng/ml flagellin (Axxora, Exeter, UK), 1 ng/ml FSL-1, 10 ng/ml lipopolysaccharide (LPS), 2 μg/ml resiquimod (R-848), 2 μM ODN2006 or 2 μM ODN2216 (Invivogen, Toulouse, France) for 18 h at 37°C, 5% CO₂. The concentrations of the TLR ligands were previously determined by titrating each ligand to determine the threshold for maximal activation.

Thwaites R.S., Unterberger S., Chamberlain G., Walker-Bone K., Davies K.A, & Sacre S. (2020). TLR1/2 and 5 induce elevated cytokine levels from rheumatoid arthritis monocytes independent of ACPA or RF autoantibody status. Rheumatology (Oxford, England), 59(11), 3533-3539.

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Stimulation and Cytokine Measurement

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Blood cell stimulation employed flat-bottom 6-well plates (TPP, Switzerland). Per well, 5 × 10⁶ cells were cultured in 3 mL Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies) supplemented with 10% fetal bovine serum (Life Technologies).
The synthetic TLR ligands Pam3Cys-SK4 (10 μg/mL), polyinosine–polycytodylic acid (polyI:C) (10 μg/mL), lipopolysaccharide (LPS) (10 μg/mL) and resiquimod (R848) (10 μg/mL), all from InvivoGen, were used for stimulation of cells over 18 h. Likewise the mitogens phorbol 12-myristate 13-acetate (PMA) (200 ng/mL), ionomycin (1 μg/mL) and concanavalin A (10 μg/mL), all from Sigma-Aldrich, were used for stimulation of cells over 18 h.
Stimulation with M. bovis (live or heat-inactivated) was done at a multiplicity of infection (MOI) of 0.1, based on preliminary experiments, for 18 h.
After 14 h, 50–100 μL of cell culture supernatant was collected and frozen for further cytokine secretion measurement (multiplex immunoassay). As soon after, Brefeldin A (10 μg/mL) (ThermoFisher) was added to the medium to block cytokine secretion; incubation was extended for another 4 h, to allow the de novo cytokine synthesis measurement (flow cytometry intracellular staining).

Démoulins T., Yimthin T., Lindtke D., Eggerschwiler L., Siegenthaler R., Labroussaa F, & Jores J. (2024). Temperature impacts the bovine ex vivo immune response towards Mycoplasmopsis bovis. Veterinary Research, 55, 18.

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