Odn m362

Manufactured by InvivoGen

Sourced in United States, France

ODN M362 is a synthetic oligodeoxynucleotide (ODN) that contains a CpG motif. It is designed to act as a toll-like receptor 9 (TLR9) agonist, which is involved in the recognition of microbial DNA and the activation of the immune system.

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CpG ODN M362 (4 citations) CpG-C (ODN M362) (2 citations)

6 protocols using odn m362

Aerosolized Pam2-ODN Immune Stimulation

Cited 1 time

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For in vivo studies, S-[2,3-bis(palmitoyloxy)-propyl]-(R)-cysteinyl-(lysyl) 3-lysine (Pam2CSK₄) and ODN M362 (InvivoGen, San Diego, CA) were reconstituted in endotoxin-free water and then diluted to the desired concentration in sterile phosphate-buffered saline (PBS). As previously described (14 (link)), the Pam2-ODN was placed in an Aerotech II nebulizer (Biodex Medical Systems, Shirley, NY) driven by 10 liters min⁻¹ air supplemented with 5% CO₂ for 20 min. The nebulizer was connected by polyethylene tubing to a polyethylene exposure chamber. Twenty-four hours prior to infections, 8 ml of Pam2 (4 µM):ODN (1 µM) was delivered via nebulization to unrestrained mice for 20 min, and then mice were returned to normal housing. For in vitro studies, Pam2-ODN was added to the culture medium 4 h prior to inoculation with virus or at the indicated time point. Pam2-ODN was used in a fixed concentration ratio but at varying doses, as indicated in the figures and Results section.

Kirkpatrick C.T., Wang Y., Leiva Juarez M.M., Shivshankar P., Pantaleón García J., Plumer A.K., Kulkarni V.V., Ware H.H., Gulraiz F., Chavez Cavasos M.A., Martinez Zayes G., Wali S., Rice A.P., Liu H., Tour J.M., Sikkema W.K., Cruz Solbes A.S., Youker K.A., Tuvim M.J., Dickey B.F, & Evans S.E. (2018). Inducible Lung Epithelial Resistance Requires Multisource Reactive Oxygen Species Generation To Protect against Viral Infections. mBio, 9(3), e00696-18.

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Breast Cancer Cell Lines and ODN Stimulation

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The breast cancer cell lines, AU565 (MAM-A⁺/HLA-A2⁺) and MCF-7 (MAM-A⁻/HLA-A2⁺), and human monocyte-like cell line, THP-1 cells, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Human CD8+T cells from HLA-A2⁺ healthy subjects were obtained from StemCell technologies (Cambridge, MA, USA). All cell cultures and incubations were performed as per provider’s recommendations and described by us before [19 (link),20 (link)]. Briefly, cells were cultured in RPMI-1640 medium at 37 °C in a 5% CO₂ incubator until they were 80% confluent. The presence of MamA and HLA-A2 expression in the breast cancer cell lines was confirmed by western blot analysis (data not shown). The ODNs—ODN2216, ODN2006, and ODN M362 (respectively controls represented by suffix ‘c’)—were at vaccigrade and obtained from InvivoGen (San Diego, CA, USA). For ODN stimulation, the breast cancer cells and the THP-1 cells were cultured in 24 well plates, 1 × 10⁵ per well pre-stimulated with PMA (phorbol myristate acetate, 10 ng/mL medium) for 16 h, washed once with RPMI-1640 media, and stimulated with ODNs (10 µM) for 5 h. These cells were later used for various experiments detailed below.

Babaer D., Amara S., McAdory B.S., Johnson O., Myles E.L., Zent R., Rathmell J.C, & Tiriveedhi V. (2019). Oligodeoxynucleotides ODN 2006 and M362 Exert Potent Adjuvant Effect through TLR-9/-6 Synergy to Exaggerate Mammaglobin-A Peptide Specific Cytotoxic CD8+T Lymphocyte Responses against Breast Cancer Cells. Cancers, 11(5), 672.

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Deciphering Immune Response Pathways

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Anti-CD11c (N418), MHC class II (I-A) (NIMR-4), anti-CD40 (HM40-3), Anti-CD80 (16-10A1), Anti-CD86 (GL1) and anti-mTLR9 (M9.D6) were obtained from eBioscience. Anti-β-actin (AC-74) and thioglycolate were from Sigma-Aldrich. Anti-p44/42 MAPK (4695), anti-phospho-p44/42 MAPK (9101), anti-p38 MAPK (9212), anti-phospho-p38 MAPK (9216), anti-IkB-α (9242), anti-phospho-IkBα (2859), anti-SAPK/JNK (9258), anti-HA (2367 and 3724) and anti-phospho-SAPK/JNK (9255) were from Cell Signaling Technology. Anti-EEA1 was from Thermo Fisher Scientific. Anti-LAMP1 (1D4B) was from abcam. Alexa Fluor488 Donkey anti-mouse IgG, Alexa Fluor488 Donkey anti-rat IgG and Alexa Fluor568 Donkey anti-rabbit IgG were from life technologies. Affinity-purified rabbit polyclonal anti-CRAMP Ab which recognizes CRAMP peptide domain was previously generated in our laboratory(38 (link)). ODN 1585, ODN 1668, ODN M362, ODN 1668-FITC, ODN M362-FITC, R848, Poly(I:C) HMW, Pam3CSK4, Zymozan, LPS-EB, pUNO-mTLR9-HA and pSELECT-puro-mcs were from invivogen. DOTAP liposomal transfection reagent (DOTAP) was from Roche applied science. Recombinant mouse CRAMP peptide (mCRAMP) was synthesized by Genemed Synthesis Inc.

Nakagawa Y, & Gallo R.L. (2014). Endogenous intracellular cathelicidin enhances TLR9 activation in dendritic cells and macrophages. Journal of immunology (Baltimore, Md. : 1950), 194(3), 1274-1284.

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Immunization Protocol for rRBCM and α-gal-BSA

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rRBCM were emulsified in Complete Freund’s Adjuvant (CFA; Incomplete Freund’s Adjuvant (IFA) plus Mycobacterium tuberculosis H37 RA; 4 mg/ml; DIFCO) with CpG (50 μg/mouse; ODN M362; Invivogen) and administered subcutaneously (s.c.). Two subsequent immunizations were emulsified in IFA+rRBC+CpG. Emulsions were administered at 200 μl per mouse, 3 times at two weeks intervals. Mice were also immunized (s.c.) with α-gal-BSA (75 μg/mouse) emulsified in CFA with two subsequent immunizations, 2 and 4 weeks thereafter with α-gal-BSA emulsified in IFA.

Yilmaz B., Portugal S., Tran T.M., Gozzelino R., Ramos S., Gomes J., Regalado A., Cowan P.J., d’Apice A.J., Chong A.S., Doumbo O.K., Traore B., Crompton P.D., Silveira H, & Soares M.P. (2014). Gut Microbiota Elicits a Protective Immune Response against Malaria Transmission. Cell, 159(6), 1277-1289.

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Aerosolized Pam2-ODN Immunostimulation

Cited 1 time

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For in vivo studies, S-[2,3-bis(palmitoyloxy)-propyl]-(R)-cysteinyl-(lysyl) 3-lysine (Pam2CSK₄) and ODN M362 (InvivoGen, San Diego, CA) were reconstituted in endotoxin-free water, then diluted to the desired concentration in sterile PBS. As previously described [16 (link)], the Pam2-ODN was placed in an Aerotech II nebulizer (Biodex Medical Systems, Shirley, NY) driven by 10 l min^-1 air supplemented with 5% CO2 for 20 min. The nebulizer was connected by polyethylene tubing to a polyethylene exposure chamber. 24 h prior to infections, 8 ml of Pam2 (4μM) -ODN (1μM) was delivered via nebulization to unrestrained mice for 20 minutes, and then mice were returned to normal housing. For in vitro studies, Pam2-ODN was added to the culture media 4 h prior to inoculation with bacteria or at the indicated time point. Pam2-ODN was given in fixed ratio, but at varying doses as indicated. For both treatments and infectious challenges, the mice are allowed to move freely and without restraint in the exposure chamber. No signs of distress have ever been observed during any of the aerosolized exposures. Aside from those that are immediately sacrificed (described below), on completion of the aerosolized exposures, mice are immediately returned to their housing cages where they have ad lib access to food and water.

Ware H.H., Kulkarni V.V., Wang Y., Pantaleón García J., Leiva Juarez M., Kirkpatrick C.T., Wali S., Syed S., Kontoyiannis A.D., Sikkema W.K., Tour J.M, & Evans S.E. (2019). Inducible lung epithelial resistance requires multisource reactive oxygen species generation to protect against bacterial infections. PLoS ONE, 14(2), e0208216.

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PRR Ligand Stimulation of Whole Blood

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Stock solutions of pattern recognition receptor (PRR) ligands (Resiquimod (R848) (Cat# tlrl-r848), ODN M362 (a class C CpG ODN, (Cat# tlrl-m362-5) and Cyclic [G(2’,5’)pA(3’,5’)p] cGAMP, (Cat# tlrl-nacga23-1) all purchased from InvivoGen, Toulouse, France) were prepared in RPMI. Fifteen µl of each 10X PRR ligand solution were distributed in 200 µl PCR tubes and stored at-20°C. On tube containing 15 µL of RPMI (Invitrogen, Cat# 31870074) without PRR ligand was used as negative control. The final concentration of the PRR ligands was R848 (1 µM), ODN M362 (5 µM) and cGAMP (20 µg/ml). Less than four hours after bleeding, heparinized blood was diluted (1/2) with RPMI before being distributed (135 µl) in the tubes containing the various stimulus. After 24 h of incubation at 37°C, the supernatants (i.e. plasma diluted with RPMI) were harvested and stored at -80°C until cytokine assays.

Congy-Jolivet N., Cenac C., Dellacasagrande J., Puissant-Lubrano B., Apoil P.A., Guedj K., Abbas F., Laffont S., Sourdet S., Guyonnet S., Nourhashemi F., Guéry J.C, & Blancher A. (2022). Monocytes are the main source of STING-mediated IFN-α production. eBioMedicine, 80, 104047.

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