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Odn 2088

Manufactured by Miltenyi Biotec
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Sourced in Germany

ODN 2088 is a synthetic oligodeoxynucleotide. It functions as a toll-like receptor 9 (TLR9) antagonist.

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Lab products found in correlation

Bafilomycin a1, by Merck Group (1 mentions) Cytochalasin d, by Merck Group (1 mentions) S1 nuclease, by Thermo Fisher Scientific (1 mentions) Rnase 3, by Thermo Fisher Scientific (1 mentions) Odn 2087, by Miltenyi Biotec (1 mentions) Ezdnase, by Thermo Fisher Scientific (1 mentions) Sb203580, by Merck Group (1 mentions) Jsh 23, by Merck Group (1 mentions) Cpg b odn 1826, by InvivoGen (1 mentions) Recombinant murine baff, by BioLegend (1 mentions) Sp2 0, by American Type Culture Collection (1 mentions) Odn 20958, by Miltenyi Biotec (1 mentions) Cytometric bead array, by BD (1 mentions) Mcc950, by Cayman Chemical (1 mentions) Chloroquine, by Merck Group (1 mentions) High molecular weight poly 1 c tlrl pic, by InvivoGen (1 mentions) 2 3 cgamp tlrl nacga23, by InvivoGen (1 mentions) Nac a7250, by Merck Group (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) Iscript cdna synthesis kit, by Bio-Rad (1 mentions)

8 protocols using odn 2088

1

Inhibition of TLR-7/8/9 in Washed Platelets

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For TLR-7/8/9 inhibition washed platelets (6 × 108 platelets/ml) were incubated in the dark with TLR7/8/9 antagonist at 2 μg/ml (ODN 2088, Miltenyi Biotec Inc.) for 30 min at 37 °C.
Zaslavsky A., Adams M., Cao X., Yamaguchi A., Henderson J., Busch-Østergren P., Udager A., Pitchiaya S., Tourdot B., Kasputis T., Church S.J., Lee S.K., Ohl S., Patel S., Morgan T.M., Alva A., Wakefield T.W., Reichert Z., Holinstat M, & Palapattu G.S. (2021). Antisense oligonucleotides and nucleic acids generate hypersensitive platelets. Thrombosis research, 200, 64-71.
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2

Scavenger Receptor Inhibitor Interactions with Nanoparticle Assemblies

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For all experiments involving inhibitors, PBMCs were preincubated with designated inhibitors for 2 h at 37 °C before the addition of nanoparticles. Internalization inhibitor concentrations used were as follows: bafilomycin A1, 100 nM; cytochalasin D, 5 μM; and all scavenger receptor inhibitors and controls, 50 μg/mL (all internalization inhibitors listed above were from Sigma-Aldrich, Saint Louis, MO). ODN 2088 was used at 10 μM (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Treated PBMCs were then tested for nanoparticle uptake or IFN induction.
To study the individual scavenger receptor inhibitors’ interactions with the assemblies, AF488-labeled NANPs (10 nM) (with and without L2K) were incubated with the various scavenger receptor inhibitors (50 μg/mL) for 30 min at room temperature. Samples were analyzed on nondenaturing native PAGE (8%, 37.5:1).
Hong E., Halman J.R., Shah A.B., Khisamutdinov E.F., Dobrovolskaia M.A, & Afonin K.A. (2018). Structure and Composition Define Immunorecognition of Nucleic Acid Nanoparticles. Nano letters, 18(7), 4309-4321.
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3

Dendritic Cell Responses to Saccharomyces cerevisiae

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pDCs and cDCs were cultured separately in 96-well flat-bottom half-area plates (Corning) at a density of 5 × 104 per well in RPMI 1640 with 5% of human serum for 48 h at 37°C with 5% of CO2, in the absence of stimuli or in the presence of R848 (InvivoGen; 100 ng/ml), SK-1 (Saccharomyces cerevisiae; MOI: 0.1–1–5–10 SK-1 cells/cDC or pDC). For the SK-1 nucleic acid stimulation experiments, the RNA and DNA (0.2 μg) were extracted from SK-1 as previously described (16 (link)), and their quality and purity were verified by agarose gel electrophoresis and using a Nanodrop 2000 spectrophotometer (Thermofisher; Supplementary Figure S6). SK-1 RNA and SK-1 DNA samples were used for pDC stimulation pre-treated with Dotap (Merck; 10 μl/μg of nucleic acids). Pre-treatment of nucleic acids with ezDNase or RNase III (Thermofisher) was performed to degrade dsDNA and dsRNA, respectively, while S1 nuclease (Thermofisher) was used to degrade ssDNA and ssRNA. For the TLR inhibition experiments, cDCs and pDCs were pre-treated for 30 min with TLR7/8 (Miltenyi Biotec ODN 2087) or TLR7/9 (Miltenyi Biotec ODN 2088) inhibitors (1 μM) and then stimulated with SK-1 (MOI 5).
Sabatini A., Guerrera G., Corsetti M., Ruocco G., De Bardi M., Renzi S., Cavalieri D., Battistini L., Angelini D.F, & Volpe E. (2022). Human Conventional and Plasmacytoid Dendritic Cells Differ in Their Ability to Respond to Saccharomyces cerevisiae. Frontiers in Immunology, 13, 850404.
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4

Murine Immune Cell Stimulation Reagents

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R848 (TLR7 ligand) and CpG-B ODN 1826 (murine TLR9 ligand) were purchased from Invivogen (San Diego, CA, USA). Lipopolysaccharide (LPS, TLR4 ligand from Salmonella Minnesota) was from Sigma-Aldrich(St. Louis, MO). Recombinant murine BAFF was from Biolegend (San Diego, CA). ODN2088 and ODN20958 were from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany). JSH23 (4-methyl-N1-(3-phenyl-propyl)-benzene-1,2-diamine, Sigma-Aldrich), SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole) (Sigma-Aldrich). Amino-actinomycin D (7-AAD) was obtained from BD Bioscience (San Jose, CA, USA). The non-secreting mouse myeloma cell line SP2/0 was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The murine anti-U1-70 K (RNP) hybridoma cell line 2.73 was a gift of Dr. Sally Hoch [17 (link)]. Additional hybridomas included 172.4 (murine anti-CD100, ATCC), 11B11 (rat IgG1 anti-mouse IL-4, ATCC), Pab101 (murine IgG2a anti-SV40 large T antigen, ATCC), 111 (murine IgG1 anti-Ku), and 162 (murine IgG2a anti-Ku).
Han S., Zhuang H., Xu Y., Lee P., Li Y., Wilson J.C., Vidal O., Choi H.S., Sun Y., Yang L.J, & Reeves W.H. (2015). Maintenance of autoantibody production in pristane-induced murine lupus. Arthritis Research & Therapy, 17, 384.
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5

Glial Cell Response to NMOSD mtDNA Stimulation

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Mixed glial cells were stimulated with DNA fractions containing abundant mtDNA. Specifically, we used DNA fractions from 100 μL CSF from NMOSD or OND patients, or the DNA fraction released from AQP4-expressing HEK293 cells stimulated with sera from NMOSD patients or healthy control subjects. After 48 h of incubation, supernatants were collected and centrifuged at 2000×g at 4 °C for 10 min.
PBMCs were pretreated with a TLR9 inhibitor, ODN2088 (4 μM, Miltenyi Biotec, Bergisch-Gladbach, Germany) and a NOD-like receptor protein 3 (NLRP3) inhibitor, and MCC950 (0.1 μM, Cayman Chemical, Ann Arbor, MI, USA) for 1 h and then treated with the DNA fraction from AQP4-expressing HEK293 cells stimulated with sera from NMOSD patients. After 10 h of incubation, supernatants were collected. IL-1β in supernatants was measured using the Cytometric Bead Array (BD Biosciences, San Jose, CA, USA).
Yamashita K., Kinoshita M., Miyamoto K., Namba A., Shimizu M., Koda T., Sugimoto T., Mori Y., Yoshioka Y., Nakatsuji Y., Kumanogoh A., Kusunoki S., Mochizuki H, & Okuno T. (2018). Cerebrospinal fluid mitochondrial DNA in neuromyelitis optica spectrum disorder. Journal of Neuroinflammation, 15, 125.
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6

Immunomodulatory ODN2088 Protocol

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Reagents used in this study were ODN2088 (130-105-815; Miltenyi Biotech), ODN2087 Ctrl (130-105-819; Miltenyi Biotech), and chloroquine (C6628; Sigma-Aldrich).
Bouvet M., Voigt S., Tagawa T., Albanese M., Chen Y.F., Chen Y., Fachko D.N., Pich D., Göbel C., Skalsky R.L, & Hammerschmidt W. (2021). Multiple Viral microRNAs Regulate Interferon Release and Signaling Early during Infection with Epstein-Barr Virus. mBio, 12(2), e03440-20.
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7

Stimulation of Innate Immune Responses

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LPS from Escherichia coli, serotype EH100 (ALX-581–010-L001), was purchased from Enzo Life Sciences. High molecular weight poly(I:C) (tlrl-pic) and 2′−3′-cGAMP (tlrl-nacga23) were purchased from Invivogen. Recombinant mouse IFNβ1 (581302) and recombinant mouse IL-10 (417-ML-005/CF) were purchased from BioLegend. ATP disodium salt (A2383), DMSO (D8418), AOAA (C13408), valinomycin (V3639), TAM (H6278) and NAC (A7250) were purchased from Sigma Aldrich. Oligomycin A from Streptomyces diastatochromogenes (M02220) was purchased from Fluorochem. FHIN1 (HY-100004), DMF (HY-17363), MMF (HY-103252), IMT1 (HY-134539) and C-178 (HY-123963) were purchased from MedChemExpress. CPG ODN 1826 (130–100-274) and ODN 2088 (130–105-815) were purchased from Miltenyi Biotec. CCCP (M20036) was purchased from Thermo Fisher.
Hooftman A., Peace C.G., Ryan D.G., Day E.A., Yang M., McGettrick A.F., Yin M., Montano E.N., Huo L., Toller-Kawahisa J.E., Zecchini V., Ryan T.A., Bolado-Carrancio A., Casey A.M., Prag H.A., Costa A.S., De Los Santos G., Ishimori M., Wallace D.J., Venuturupalli S., Nikitopoulou E., Frizzell N., Johansson C., Von Kriegsheim A., Murphy M.P., Jefferies C., Frezza C, & O’Neill L.A. (2023). Macrophage fumarate hydratase restrains mtRNA-mediated interferon production. Nature, 615(7952), 490-498.
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8

Splenic B Cell Activation Assay

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Naive splenic B cells were purified using the B cell isolation kit (Miltenyi Biotech) according to the manufacturer's protocol. Purified B cells (purity 90%-92%) were then incubated either alone, with different ratios of parasites, or with TLR agonists: 3 mg/ml CpG (InvivoGen), 5 mg/ml R837 (InvivoGen), 3 mg/ml poly (I:C) (Amersham), and 10 mg/ml LPS (Sigma). B cells were also incubated with 1 mg/ml ODN2088 (Miltenyi) and a TLR7 and 9 inhibitor (Stunz et al., 2002) . Cells were cultured in supplemented DMEM (Gibco, Invitrogen). For cytokine mRNA expression analysis, confocal microscopy, and immunoblots, cells were incubated for 8 or 24 hr at 37 C.
Quantitative Real-Time PCR RNA from isolated B cells from in vitro or in vivo experiments was extracted using the RNeasy mini kit (QIAGEN) as described by the manufacturer. Reverse transcription was carried out using the iScript cDNA synthesis kit (Bio-Rad) following the manufacturer's protocol. Real-time PCR analysis was performed using iTaq Universal SYBR Green Supermix (Bio-Rad) . The IL-1a, IL-1b, IL-6, IL-10, IFN-a, IFN-b, and HPRT genes were amplified using primers whose sequences are described below. All PCRs were carried out with the Stratagene mx3005p real-time PCR system. Data were normalized to HPRT and expressed as fold increase to naive controls.
Silva-Barrios S., Smans M., Duerr C.U., Qureshi S.T., Fritz J.H., Descoteaux A, & Stäger S. (2016). Innate Immune B Cell Activation by Leishmania donovani Exacerbates Disease and Mediates Hypergammaglobulinemia. Cell reports, 15(11).
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