Cpg oligonucleotide

Manufactured by InvivoGen

Sourced in United States

CpG oligonucleotide is a synthetic DNA fragment that contains unmethylated CpG motifs. CpG oligonucleotides are used as a tool in research to activate the immune system by binding to Toll-like receptor 9 (TLR9).

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Lab products found in correlation

Flagellin, by InvivoGen (2 mentions) Cytofix cytoperm kit, by BD (2 mentions) Facscanto 2 flow cytometer, by BD (2 mentions) Golgistop, by BD (2 mentions) Golgiplug, by BD (2 mentions) Ionomycin, by Merck Group (2 mentions) Phorbol myristate acetate (pma), by Merck Group (2 mentions) Turbofect, by Thermo Fisher Scientific (2 mentions) Diphtheria toxoid, by Statens Serum Institut (1 mentions) Pokeweed mitogen, by Merck Group (1 mentions) Pvdf 96 well plates, by Merck Group (1 mentions) Nigericin, by InvivoGen (1 mentions) Anti phospho p38 t180 y182, by Cell Signaling Technology (1 mentions) Trizol reagent 15596018, by Thermo Fisher Scientific (1 mentions) Bapta am b1205, by Thermo Fisher Scientific (1 mentions) Anti chmp4b 13683 1 ap, by Proteintech (1 mentions) Anti β actin sc 1615, by Santa Cruz Biotechnology (1 mentions) Anti caspase 1 ag 20b 0042, by Adipogen (1 mentions) Ultrapure lps, by InvivoGen (1 mentions) Anti ly6c pe, by BioLegend (1 mentions)

Close spelling variants of this product

CpG oligonucleotide (ODN1826), (3 citations) Class‐A CpG oligonucleotide ODN‐2336 (CpGA, (3 citations) CpG oligonucleotide 1826 (3 citations) Class B CpG oligonucleotide (3 citations) Class‐A CpG oligonucleotide ODN‐2336 (2 citations) CpG oligonucleotide 1668 (2 citations) CpG oligonucleotides ODN 1826 (2 citations)

12 protocols using cpg oligonucleotide

Memory B Cell Assessment via Cultured ELISpot

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PVDF 96 well plates (Millipore) were coated with 100 μl of either 5 μg/ml (capsular group A and C) of purified meningococcal polysaccharide (National Institute for Biological Standards and Control (NIBSC) 98/722 and 07/318) conjugated to 5 μg/ml methylated human albumin (NIBSC), 10 μg/mL diphtheria toxoid (Statens Serum Institut 2675) or phosphate-buffered saline (background control). Prior to cells being seeded onto the plates, all wells were blocked with complete medium.
Memory B cells were assessed using cultured ELISpot performed on blood samples collected at days 0, 28 and 56. PBMCs were suspended in R10 at a concentration of 2 × 10⁶ cells/ml. These cells were cultured with an additional 100 μl of RPMI with 10% newborn bovine serum (NBBS), Staphylococcus aureus Cowan strain (SAC) at 1:2500 dilution of the Pansorbin cell suspension (Calbiochem-Novabiochem), 166 ng/ml pokeweed mitogen (Sigma-Aldrich) and 3.4 μg/ml CpG oligonucleotide (InvivoGEN). The cells were incubated for 6 days at 37 °C in 5% carbon dioxide and 95% humidity, after which time they were washed and processed as described by Lazarus et al. [9 (link)].

O’Connor D., Clutterbuck E.A., Thompson A.J., Snape M.D., Ramasamy M.N., Kelly D.F, & Pollard A.J. (2017). High-dimensional assessment of B-cell responses to quadrivalent meningococcal conjugate and plain polysaccharide vaccine. Genome Medicine, 9, 11.

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Immune Cell Activation Assay

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Ultrapure LPS, flagellin, nigericin, CpG oligonucleotide, and poly(dA:dT) were purchased from InvivoGen. Silica (MIN-U-SIL 15) was obtained from US Silica. ATP was from Sigma-Aldrich. Pam3Cys was from EMC Microcollections. X-tremeGENE HP DNA transfection reagent was from Roche. Transfection reagent Profect P1 was from Targeting Systems. Acridine Orange (L13159) was from Alfa Aesar. TRIzol reagent (15596018), BAPTA-AM (B1205), and Alexa Fluor 594-labeled zymosan particles (Z23374) were from Thermo Fisher Scientific. Antibodies for immunoblotting include anti-CHMP4B (13683-1-AP) from Proteintech; anti-IKKα (05-536) from Millipore; anti-Mcu (14997), anti-phospho-IkBα (S32), anti-phospho-IKKα/β (S176/180), anti-phospho-p65(S536), anti-p65, anti-phospho-ERK1/2 (T202/Y204), anti-phospho-JNK (T183/Y185), and anti-phospho-p38 (T180/Y182) from Cell Signaling Technology; anti-β-actin (sc-1615) from Santa Cruz Biotechnology; anti-NLRP3 (Cryo-2), anti-Asc (AL177), and anti-caspase-1 (AG-20B-0042) from Adipogen; and anti-IL-1β (AF-401-NA) from R&D Systems. Antibodies for the flow cytometry assay include anti-CD45-PE Cy7 (103114), anti-CD11b-FITC (101206), anti-Ly-6G-PB (127612), and anti-Ly-6C-PE (128008) from BioLegend.

Dong H., Zhao B., Chen J., Liu Z., Li X., Li L, & Wen H. (2022). Mitochondrial calcium uniporter promotes phagocytosis-dependent activation of the NLRP3 inflammasome. Proceedings of the National Academy of Sciences of the United States of America, 119(26), e2123247119.

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Cytokine production by TLR agonists

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PBMCs were cultured alone or with various TLR agonists including lipoteichoic acid (LTA 1 μg/mL, TLR2 ligand), Poly I:C (50 μg/mL, TLR3 ligand), E. coli lipopolysaccharide (LPS 10 ng/mL, TLR4 ligand), Flagellin (1 μg/mL, TLR5 ligand), Imiquimod (10 μg/mL, TLR7 ligand), Gardiquimod (10 μg/mL, TLR8 ligand) or CpG oligonucleotide (CpG 3 μg/mL, TLR9 ligand); all purchased from InvivoGen, CA, United States. All cultures were plated in duplicate in 96-well round-bottom plates in 250 μL RPMI (Gibco, Life Technology, Grand Island, NY, United States) supplemented with 10% foetal calf serum (Australia Biosearch, Australia) and incubated at 37 °C with 5% CO₂ for 24 h (LTA, Poly I:C, LPS and Flagellin) or 48 h (Imiquimod, Gardiquimod or CpG). The supernatants were then removed and stored at -20 °C until cytokine analysis.

Baird A.C., Mallon D., Radford-Smith G., Boyer J., Piche T., Prescott S.L., Lawrance I.C, & Tulic M.K. (2016). Dysregulation of innate immunity in ulcerative colitis patients who fail anti-tumor necrosis factor therapy. World Journal of Gastroenterology, 22(41), 9104-9116.

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Stimulation and Detection of Cytokine-Producing B Cells

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Aliquots of 10⁶ PBMC were resuspended in 1 mL of Complete Medium with 50 ng/mL Phorbol 12‐myristate 13‐acetate (PMA) (Sigma‐Aldrich, St. Louis, MO) and 750 ng/mL Ionomycin (Sigma‐Aldrich) in presence of 2 μg/mL Brefeldin A (GolgiPlug, BD Biosciences) and 2.1 μmol/L Monensin (GolgiStop, BD Biosciences) in polypropylene tubes, and incubated for 4 h at 37°C in 5% CO₂. To identify IL‐10‐producing B cells, PBMC were preincubated with 3 μg/mL of CpG oligonucleotide (InvivoGen, San Diego, CA) during 20 h at 37°C with 5% CO₂ prior to stimulation. After incubation, PBMC were washed with PBS and stained for 30 min at 4°C in the dark with appropriate amounts of monoclonal antibodies recognizing the surface antigens. Then, cells were washed with PBS, fixed, and permeabilized for 20 min at 4°C in the dark with Cytofix/Cytoperm Kit (BD Biosciences), washed twice with Perm/Wash solution (BD Biosciences), and stained intracellularly 30 min at 4°C in the dark with monoclonal antibodies recognizing the following cytokines: IFN‐gamma, TNF‐alpha, IL‐17, and IL‐10. After two washes, PBMC were analyzed in a FACSCanto II flow cytometer (BD Biosciences).

Medina S., Sainz de la Maza S., Villarrubia N., Álvarez‐Lafuente R., Costa‐Frossard L., Arroyo R., Monreal E., Tejeda‐Velarde A., Rodríguez‐Martín E., Roldán E., Álvarez‐Cermeño J.C, & Villar L.M. (2019). Teriflunomide induces a tolerogenic bias in blood immune cells of MS patients. Annals of Clinical and Translational Neurology, 6(2), 355-363.

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Influenza Subunit and Split Vaccines Evaluation

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The influenza virus subunit vaccines were provided by Dr. Na Gyong Lee (Sejong University, Seoul, Korea). The trivalent HA vaccine comprised the HA subunits from three influenza virus strains: A/New Caledonia/20/99 (H1N1), A/Panama/2007/99 (H3N2) and B/Shandong/7/97. The split H1N1 influenza virus vaccine was provided by Green Cross Co. (Yongin, Gyeonggi, Korea). The vaccine comprised the split influenza A/California/7/2009 (NYMCX-181) (H1N1) virus. Various adjuvants were used including cholera toxin (CT; List Biological Laboratories, Campbell, CA), polyinosinic-polycytidylic acid (poly(I:C), monophosphoryl lipid A (MPLA) from Salmonella minnesota, CpG oligonucleotide (InvivoGen, San Diego, CA), and ImjectAlum (Thermo Scientific, Rockford, IL).

Kim E.D., Han S.J., Byun Y.H., Yoon S.C., Choi K.S., Seong B.L, & Seo K.Y. (2015). Inactivated Eyedrop Influenza Vaccine Adjuvanted with Poly(I:C) Is Safe and Effective for Inducing Protective Systemic and Mucosal Immunity. PLoS ONE, 10(9), e0137608.

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Multiparametric Flow Cytometry Analysis

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To study surface antigens, cells were stained with the respective monoclonal antibodies during 30 min at 4°C in the dark, washed with saline and analyzed in a FACSCanto II flow cytometer (BD Biosciences), as described before (12 (link)).
For intracytoplasmic cytokine detection, cells were stimulated with phorbol-12-myristate-13-acetate (PMA, Merck) and Ionomycin (Merck), in presence of Brefeldin A (GolgiPlug, BD Biosciences) and Monensin (GolgiStop, BD Biosciences), and then incubated 4 h at 37°C in 5% CO2 atmosphere. For the analysis of IL-10 producing B cells, PBMC were incubated prior to stimulation with CpG oligonucleotide (InvivoGen) for 20 h. After incubation, surface markers were stained, then the cellular membrane permeabilized with Cytofix/Cytoperm Kit (BD Biosciences), and incubated with intracellular antibodies, following the same protocol previously described (12 (link)).
Cells were analyzed using FACSDiva Software V.8.0 (BD Biosciences). A minimum amount of 5x10 (4 (link)) events were acquired. Gating strategy is defined in Figure 1.

Walo-Delgado P.E., Monreal E., Medina S., Quintana E., Sainz de la Maza S., Fernández-Velasco J.I., Lapuente P., Comabella M., Ramió-Torrentà L., Montalban X., Midaglia L., Villarrubia N., Carrasco-Sayalero A., Rodríguez-Martín E., Roldán E., Meca-Lallana J., Alvarez-Lafuente R., Masjuan J., Costa-Frossard L, & Villar L.M. (2021). Role of B Cell Profile for Predicting Secondary Autoimmunity in Patients Treated With Alemtuzumab. Frontiers in Immunology, 12, 760546.

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Humanized Mice for GVHD Studies

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NOD-Scid-IL-2Rγ^null (NSG) mice (Jackson laboratory, Bar Harbor, Michigan, USA) were bred in the animal facility of St-Louis Research Institute and housed under specific pathogen-free conditions. Eight- to 10-week-old female mice were irradiated (1.3 Gy) 24 hours prior to injection of 5×10⁶ human PBMCs (huPBMCs) in the caudal vein. Development of GVHD was monitored three times per week based on weight loss, hunching posture, reduced mobility and hair loss. Human chimerism in peripheral blood (per cent of human CD45⁺ cells) was assessed weekly. When indicated, mice received 1 nmol 5-OP-RU and/or 100 ng human IL-15 and/or 10 µg CpG Oligonucleotide (class B ODN, Invivogen) intraperitoneally (i.p) every 2 days from the day of PBMCs infusion. Mice were sacrificed at the indicated time, or when weight loss was >15%. Peripheral blood, spleen, liver, lungs and intestine were harvested, and cells were isolated as described in online supplemental methods.

Tourret M., Talvard-Balland N., Lambert M., Ben Youssef G., Chevalier M.F., Bohineust A., Yvorra T., Morin F., Azarnoush S., Lantz O., Dalle J.H, & Caillat-Zucman S. (2021). Human MAIT cells are devoid of alloreactive potential: prompting their use as universal cells for adoptive immune therapy. Journal for Immunotherapy of Cancer, 9(10), e003123.

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Stimulation of 3T6 Cells with Innate Immune Agonists

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Polyinosinic:polycytidylic acid (GE Healthcare) was used to stimulate 3T6 cells; specifically, 2 × 10⁶ cells were transfected by TurboFect (Thermo Fisher Scientific) with 20 μg of poly (I:C). The cells were incubated for 16 h and then collected for isolation of RNA or for the preparation of cell lysates. c‐di‐GAMP (InvivoGen, San Diego, CA, USA), pDNA, CpG oligonucleotide (InvivoGen) or 26‐mer DNA (G3‐YSD, InvivoGen) were used to stimulate 3T6 cells as follows: cells (4 × 10⁶) were transfected with 4 μg of c‐di‐GAMP, 6 μg of pDNA, or 4 μg CpG by Amaxa nucleofector (Lonza), incubated for a further 6 h, and collected for the isolation of RNA.

Ryabchenko B., Soldatova I., Šroller V., Forstová J, & Huérfano S. (2021). Immune sensing of mouse polyomavirus DNA by p204 and cGAS DNA sensors. The Febs Journal, 288(20), 5964-5985.

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Evaluating Immunomodulatory Effects of Antimalarial Drugs

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PBMCs were isolated from the samples using Ficoll-Paque PLUS (GE Healthcare, Uppsala, Sweden). Isolated PBMCs were plated into cell culture plates containing 1.5 ml of RPMI 1640 (1×) [+] l-glutamine [−] phenol red medium (Gibco Life Technologies, Grand Island, NY) conditioned with 10% fetal bovine serum and 1% penicillin-streptomycin in the amount of 2.5 × 10⁶ PBMCs per well. The PBMCs were subsequently treated with 3 µmol/L concentrations of HCQ (Sigma-Aldrich, St. Louis, MO), QC dihydrochloride (Sigma-Aldrich), and a combination QC and HCQ in the appropriate wells and allowed to incubate for 18 hours at 37 °C and5%CO₂. To also test the effect of these drugs on stimulated versus unstimulated PBMCs, 3-µmol/L concentrations of Escherichia coli-derived LPS (Sigma-Aldrich) and CpG oligonucleotides (InvivoGen, San Diego, CA) were used to stimulate secretion of TNF-α and IFN-α, respectively.

Alves P., Bashir M.M., Wysocka M., Zeidi M., Feng R, & Werth V.P. (2017). Quinacrine Suppresses Tumor Necrosis Factor-α and IFN-α in Dermatomyositis and Cutaneous Lupus Erythematosus. The journal of investigative dermatology. Symposium proceedings, 18(2), S57-S63.

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Immune Response Modulation Mechanisms

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LPS (from Escherichia coli serotype 055:B5) and poly(I:C) was purchased from Sigma-Aldrich. CpG oligonucleotides were purchased from InvivoGen (San Diego, CA). Recombinant human IFN-α, IFN-β, IFN-γ, GM-CSF, IL-4 and TNF-α were purchased from PeproTech. JAK inhibitor pyridone 6 (P6) was obtained from Sigma-Aldrich. PI3-kinase inhibitor LY294002, extracellular signal-regulated kinase (ERK) inhibitor PD98059 and p38 MAPK inhibitor SB202190 were obtained from Calbiochem (La Jolla, CA). The primary antibodies against STAT3, IFNAR1 and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The primary antibodies against STAT1, AKT, IRF3 and IRF7 were purchased from Cell Signalling Technology (Beverly, MA). The neutralizing antibody against IFN-α and that against IFN-β were purchased from PeproTech. The primary antibody against ATX was produced by our lab as described previously [33 (link)].

Song J., Guan M., Zhao Z, & Zhang J. (2015). Type I Interferons Function as Autocrine and Paracrine Factors to Induce Autotaxin in Response to TLR Activation. PLoS ONE, 10(8), e0136629.

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