Cpg odn 2395

Manufactured by InvivoGen

Sourced in United States

CpG ODN 2395 is a synthetic oligodeoxynucleotide (ODN) that contains unmethylated CpG motifs. It functions as a Toll-like receptor 9 (TLR9) agonist, stimulating the innate immune response.

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Lab products found in correlation

Imiquimod, by InvivoGen (4 mentions) 2 3 cgamp, by InvivoGen (2 mentions) Addavax, by InvivoGen (2 mentions) Addas03, by InvivoGen (2 mentions) Flagellin, by InvivoGen (2 mentions) Zymosan, by InvivoGen (2 mentions) Pam3csk4, by InvivoGen (2 mentions) Dotap, by Roche (2 mentions) Pam2csk4, by InvivoGen (2 mentions) Cantell strain, by Charles River Laboratories (1 mentions) High molecular weight poly 1 c, by InvivoGen (1 mentions) Golgiplug, by BD (1 mentions) Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions) Gm csf, by Kingfisher Biotech (1 mentions) M csf, by Kingfisher Biotech (1 mentions) Ficoll paque plus, by GE Healthcare (1 mentions) Celecoxib, by R&D Systems (1 mentions) Cpg odn 2006, by InvivoGen (1 mentions) Ns 398, by R&D Systems (1 mentions) Resiquimod, by Merck Group (1 mentions)

25 protocols using cpg odn 2395

Evaluating COBRA HA and N1-I NA Vaccines in Mice

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All procedures were reviewed and approved by the University of Georgia Institutional Animal Care and Use Committee (IACUC). For the H1 HA study, 10 six-to-eight-week-old male and female BALB/c mice (Charles River Laboratories, Wilmington, MA, USA) per group were immunized subcutaneously with 20 μg of Y2 COBRA HA, CA09 HA, or phosphate-buffered saline (PBS) adjuvanted with AddaVax, AddaS03, CpG ODN 2395, and Alhydrogel (Invivogen, San Diego, CA, USA), or with PBS as a no adjuvant control. Mice were bled at 27 days post-prime for d27 serum, then immunized at 28 days post-prime with 20 μg of the antigen. At 56 days post-prime, animals were bled for d56 serum, and then euthanized with Avertin.
For the N1-I NA study, five six-to-eight-week-old female BALB/c mice (Charles River Laboratories, Wilmington, MA, USA) per group were immunized intramuscularly with 6 μg of N1-I COBRA HA or PBS adjuvanted with AddaVax, AddaS03, CpG ODN 2395, and Alhydrogel (Invivogen, San Diego, CA, USA), or with PBS as a no adjuvant control. Mice were bled at 27 days post-prime for d27 serum, then immunized at 28 days post-prime with 6 μg of the antigen. At 56 days post-prime, animals were bled for d56 serum, and then euthanized with Avertin.

Nagashima K., Abbadi N., Vyas V., Roegner A., Ross T.M, & Mousa J.J. (2023). Adjuvant-Mediated Differences in Antibody Responses to Computationally Optimized Hemagglutinin and Neuraminidase Vaccines. Viruses, 15(2), 347.

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Innate Immune Response Profiling

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Patient and control PBMCs were thawed and seeded in RPMI supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin (cRPMI) at a density of 5 × 10⁵ cells/well for infection experiments and 1 × 10⁶ cells/well for stimulation with 2′3′-cGAMP and dsDNA and incubated overnight at 37 °C and 5% CO₂. Cells were infected with 50 HAU of Sendai virus (SeV) (Cantell strain, Charles River), 3 MOI of HSV1 (KOS Strain), or 0.2 MOI of cell-free (CF) VZV debris (rOKa strain) or CF debris mock). Following 24 h of infection, supernatants were harvested for mesoscale, and cells were lysed for RNA isolation. In addition, PBMCs were stimulated with 100 µg/mL of 2′3′-cGAMP (InvivoGen) or 2 µg/mL of transfected ht (herring testes) dsDNA for 3 h following cell lysis for Western blotting or stimulated with 50 µg/mL high molecular weight poly(I:C) (InvivoGen, USA) or 5 µg/mL CpG ODN2395 (InvivoGen, USA) for 6 h before being lysed for downstream RT-qPCR analysis.

Thomsen M.M., Skouboe M.K., Møhlenberg M., Zhao J., de Keukeleere K., Heinz J.L., Werner M., Hollensen A.K., Lønskov J., Nielsen I., Carter-Timofte M.E., Zhang B., Mikkelsen J.G., Fisker N., Paludan S.R., Assing K, & Mogensen T.H. (2024). Impaired STING Activation Due to a Variant in the E3 Ubiquitin Ligase AMFR in a Patient with Severe VZV Infection and Hemophagocytic Lymphohistiocytosis. Journal of Clinical Immunology, 44(2), 56.

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Immune Response to TLR Ligands in Whole Blood

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Within a maximum of 4 h after drawing, 100 µl of heparinized WB samples were incubated 4 h with the following TLR ligands: heat-killed Listeria monocytogenes (HKLM, TLR2-L, 10⁸ HKLM/ml), Poly(I:C) (TLR3-L, 100 µg/ml), CL097 (imidazoquinoline compound, TLR7/8-L, 2 µg/ml) and CPG ODN2395 [Type C CPG oligonucleotide, TLR9-L, 50 µM, all obtained from Invivogen (Toulouse, France)], or lipopolysaccharides (from Escherichia coli O26:B6, TLR4-L, 0.1 µg/ml) purchased from Sigma-Aldrich (St. Louis, MI, USA). GolgiPlug (BD Biosciences, Le Pont de Claix) was added during the last 3 h of incubation to inhibit cellular cytokine release. Incubation in medium alone served as a negative control condition.

Braudeau C., Néel A., Amouriaux K., Martin J.C., Rimbert M., Besançon A., Giraudet S., Terrien C., Aliaga M., Salabert-Le Guen N., Hémont C., Hamidou M, & Josien R. (2017). Dysregulated Responsiveness of Circulating Dendritic Cells to Toll-Like Receptors in ANCA-Associated Vasculitis. Frontiers in Immunology, 8, 102.

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Bone Marrow Cell Activation and Cytokine Measurement

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Bone marrow cells were cultured in RPMI (Invitrogen, cat#11965118) with 10% FBS, 2mM L-glutamine, 1mM Sodium pyruvate, 10mM HEPES buffer, 1% Non-essential amino acids, 50μM 2-Mercaptoethanol, 1% Pen/Strep, 20ng/ml G M-CSF (Kingfisher, cat# RP0407M) or 20ng/ml M-CSF (Kingfisher, cat# RP0462M). The medium was changed at day 3 and 6. At day 6, cells (1×10⁶) were transferred to a 24-well plate with fresh medium. Cells were activated at day 7 with 10μg/ml CDA, CDG, 2′3′-cGAMP or 5μg/ml Rp-Rp-ssCDA in culture directly. Mouse IFNβ was measured in culture supernatant after 5hrs by ELISA (PBL Bioscience, cat#42410). Separately, BMDM and BMDC were activated with 5μg/ml HSV DNA (Invivogen, cat# tlrl-hsv60n) and Vaccinia virus DNA (Invivogen, cat# tlrl-vav70n) transfected with lipofectamine®2000(27 (link)) and mouse IFNβ was measured in culture supernatant after 5hrs by ELISA. Alternatively, BMDC were activated with Heat kill streptococcus pneumonia (HKSP) (10⁸c.f.u/ml) (Invivogen, cat# tlrl-hksp), LPS from Salmonella (25ng/ml) (Sigma, cat# L7261), Imiquimod (4ng/ml) (Invivogen, cat# tlrl-imqs) or CpG-ODN2395 (8ng/ml) (Invivogen, cat# tlrl-2395). Mouse TNFα and IFNβ were measured in culture supernatant after 5hrs by ELISA.

Patel S., Blaauboer S.M., Tucker H.R., Mansouri S., Ruiz-Moreno J.S., Hamann L., Schumann R.R., Opitz B, & Jin L. (2016). The common R71H-G230A-R293Q (HAQ) human TMEM173 is a null allele. Journal of immunology (Baltimore, Md. : 1950), 198(2), 776-787.

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Mucosal Immune Responses to Gp140-F Env

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Two experimental groups of animals were inoculated three times with soluble gp140-F Env trimers in combination with 75 μg AbISCO (Isconova AB, now Novavax) for one group, or 75 μg AbISCO with 500 μg CpG ODN2395 (Invivogen) to the other group. Env protein was given at 200 μg per animal for the first inoculation and 100 μg for the following injections. The control group was inoculated with 75 μg AbISCO and 500 μg CpG ODN2395 only. We selected this dose of CpG based upon previous studies in rhesus macaques51 (link) and humans4 (link). Inoculations were performed at weeks 0, 8 and 32 by the i.m. route of injection, given in a total volume of 1 ml, divided equally between the left and right hind leg. Blood samples were taken at eight different times, vaginal and rectal samples were taken at four different times and bone marrow samples were taken at three different time points (Figure 1A). Blood, bone marrow and lavages were sampled as previously described17 (link)52 (link). PBMCs were separated from EDTA blood by Ficoll-Paque™ PLUS (GE Healthcare). Bone marrow cells were treated with red-blood cell lysis and frozen in FCS in 10% DMSO at −150°C.

Martinez P., Sundling C., O'Dell S., Mascola J.R., Wyatt R.T, & Karlsson Hedestam G.B. (2015). Primate immune responses to HIV-1 Env formulated in the saponin-based adjuvant AbISCO-100 in the presence or absence of TLR9 co-stimulation. Scientific Reports, 5, 8925.

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Lipoxin B4 Modulates Memory B Cell Activation

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Purified CD19⁺ B cells or PBMCs were cultured in RPMI 1640 (GIBCO/Invitrogen, Carlsbad, CA) supplemented with 5% fetal bovine serum, 2mM L-glutamine, 5×10⁻⁵M 2-mercaptoethanol, 10mM HEPES and 50μg/ml gentamicin. The memory B cell-inducing cocktail was composed of PWM (pokeweed mitogen) (1:100,000, generous gift from Dr. Shane Crotty, La Jolla Institute for Allergy & Immunology), SAC (protein A from Staphylococcus aureus) (1:10,000, Sigma Aldrich, Saint Louis, MO) and CpG ODN2395 (0.5μg/ml, Invivogen, San Diego, CA). CpG ODN2006 (0.5μg/ml) was from Invivogen, IL-2 (10ng/ml) was from R&D Systems, and Resiquimod (R848, 1μg/ml) was from Sigma. Lipoxin B₄ (LXB_4, 5S, 14R, 15S- trihydroxy- 6E, 8Z, 10E, 12E- eicosatetraenoic acid) (Cayman Chemical Company, Ann Harbor, MI) was suspended in ethanol and supplemented in cell culture at nanomolar concentrations. Vehicle control was defined as 1x PBS with 0.03% ethanol by volume, equivalent to the highest concentration of LXB₄ used. Cells were pretreated with either vehicle control or LXB₄ for 30 minutes, then were treated with the memory B cell-inducing activators or left unstimulated. Additional SPM treatments were added every 24 hours for the duration of the experiment. Celecoxib and NS-398 (R&D system, Minneapolis, MN) were dissolved in DMSO and added to cell culture at the indicated concentrations.

Kim N., Lannan K.L., Thatcher T.H., Pollock S.J., Woeller C.F, & Phipps R.P. (2018). Lipoxin B4 (LXB4) enhances human memory B cell antibody production via upregulating cyclooxygenase-2 (COX2) expression. Journal of immunology (Baltimore, Md. : 1950), 201(11), 3343-3351.

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Pattern Recognition Receptor Ligand Study

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All chemicals used in the study were of cell culture grade and endotoxin-free. Pattern recognition receptor ligands used in this study were purchased from InvivoGen (LPS, Pam₃CSK₄, CpG ODN 2395, zymosan, MDP and flagellin) and curdlan, laminarin and S.c. mannan from Sigma-Aldrich. All samples were prepared in endotoxin-free water. The endocytosis inhibitor Cyt D was purchased from Sigma-Aldrich, the transfection reagent DOTAP from Roche and DNase I from Invitrogen. The chitinase inhibitor Bisdionin C was a kind gift from I. Eggleston (Bath).

Wagener J., Malireddi R.K., Lenardon M.D., Köberle M., Vautier S., MacCallum D.M., Biedermann T., Schaller M., Netea M.G., Kanneganti T.D., Brown G.D., Brown A.J, & Gow N.A. (2014). Fungal Chitin Dampens Inflammation through IL-10 Induction Mediated by NOD2 and TLR9 Activation. PLoS Pathogens, 10(4), e1004050.

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Immunomodulatory Pathway Activation Assay

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CpG ODN2395, TLR9 inhibitors ODN2088, and CQ were purchased from InvivoGen (San Diego, CA, USA). Recombinant TNF-α was purchased from Abcam (Cambridge, UK). All MAPK inhibitors were purchased from Calbiochem (Darmstadt, Germany). MAPK antibodies against ERK, phospho-ERK (p-ERK), p38, phospho-p38 (p-p38), JNK, and phospho-JNK (p-JNK) were from Cell Signaling Technology (Beverly, MA, USA). Dulbecco’s modified Eagle’s medium (DMEM), RPMI-1640, fetal bovine serum (FBS), and antibiotics for cell culture were purchased from Invitrogen (Carlsbad, CA, USA).

Jeon J.H., Kim Y.H., Choi M.K., Kim K.A., Lee H.R., Jang J., Kim Y.R., Chun J.H., Eo S.K., Kim T.S, & Rhie G.E. (2014). Bacillus anthracis genomic DNA enhances lethal toxin–induced cytotoxicity through TNF-α production. BMC Microbiology, 14, 300.

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Immunological Responses in TRIM21-Deficient Mice

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All animal work was licensed under the UK Animals (Scientific Procedures) Act 1986 and approved, together with human cell work, by the Medical Research Council Animal Welfare and Ethical Review Body. C57BL/6 WT (T21^+/+) and TRIM21-deficient mice (T21^−/−) were obtained from Jackson Laboratories. Ad5 injections at the indicated dose were administered in 100 µL endotoxin-free PBS by lateral tail vein injection (i.v.) or in 20 µL by intranasal administration. PR8 infections were by intranasal administration of 10³ pfu PR8-SL8 in 20 µL endotoxin-free PBS (Teknova) 10 d after immunization with Ad5-Ova. Details of PR8-SL8 are provided in SI Appendix, Supplementary Materials and Methods. For challenge with TLR agonists, mice were injected with 50 µg of resiquimod (R848; Invivogen) and 50 µg CpG (ODN 2395; Invivogen) in 100 µL endotoxin-free water and culled 4 h post injection.

Bottermann M., Foss S., van Tienen L.M., Vaysburd M., Cruickshank J., O’Connell K., Clark J., Mayes K., Higginson K., Hirst J.C., McAdam M.B., Slodkowicz G., Hutchinson E., Kozik P., Andersen J.T, & James L.C. (2018). TRIM21 mediates antibody inhibition of adenovirus-based gene delivery and vaccination. Proceedings of the National Academy of Sciences of the United States of America, 115(41), 10440-10445.

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Cytokine expression analysis in mouse and human cells

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Mouse and human cell suspensions were incubated with LPS EB-ultrapure from E. coli O111:B4 (1 μg/mL, In vivogen) or CpG-ODN 2395 (5μM, Invivogen) at 37°C for 4 hours in presence of Golgi stop and Golgi plug (BD Biosciences) and then assessed for cytokine expression by intracellular staining.

Bourdely P., Petti L., Khou S., Meghraoui-Kheddar A., Elaldi R., Cazareth J., Mossadegh-Keller N., Boyer J., Sieweke M.H., Poissonnet G., Sudaka A., Braud V.M, & Anjuère F. (2022). Autofluorescence identifies highly phagocytic tissue-resident macrophages in mouse and human skin and cutaneous squamous cell carcinoma. Frontiers in Immunology, 13, 903069.

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