Computational protocol: Guanine-Modified Inhibitory Oligonucleotides Efficiently Impair TLR7- and TLR9-Mediated Immune Responses of Human Immune Cells

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Protocol publication

[…] Murine pDCs, human PBMCs or B-cells were stimulated with the TLR9 agonists CpG-ODN 2006 or 2216 or TLR7 agonists ORN 22075 (R-1075, C*C*G*U*C*U*G*U*U*G*U*G*U*G*A*C*U*C) [], imiquimod or R848 in the presence of 10-fold titrated amounts of INH-ODNs (0.01–10 μM). The medium used was RPMI supplemented with 10% FCS, 100 IU/ml penicillin, 100 μg/ml streptomycin (PAA Laboratories GmbH, Pasching, Austria) and in case of murine cells 50 μM 2-ME. Cytokine levels in the supernatant were determined after 24 hours to 6 days of culture in 96 well microtiterplates (Falcon, Colorado, USA).Determination of cytokines, intracellular ATP- and extracellular LDH-levels Human IL-6 (R&D Systems Europe, Ltd., United Kingdom) and human IFN-α (eBioscience, Frankfurt, Germany) were determined using commercially available ELISA kits. Murine IFN-α was measured using antibodies from tebu-bio GmbH (Offenbach, Germany) and Jackson Immuno Research Europe ltd. (Suffolk, United Kingdom). The assays were performed according to the manufacturer’s manual. Intracellular ATP-levels were measured by CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, USA). Briefly, cells were washed and subsequently lysed with CellTiter-Glo Buffer. The ATP-content of the lysate was measured via luminometer (Berthold Titertek Instruments, Pforzheim, Germany). Extracellular LDH-levels were determined using CytoTox 96 Non-Radioactive Cytotoxicity Assay. Data were analyzed using SigmaPlot 12.0 (Systat Software, USA). [...] PBMCs were stained with CD20, CD86 and HLA-DR antibodies. CD20+ cells were assayed for CD86 and HLA-DR expression. Flow cytometry was performed with a FACS Calibur instrument (BD Biosciences, San Jose, CA, USA) or a CyAn ADP9 color device (Beckman Coulter, Krefeld, Germany), the data were analysed using the FlowJo software (Tree Star Inc, OR, USA). […]

Pipeline specifications

Software tools SigmaPlot, FlowJo
Applications Miscellaneous, Flow cytometry
Organisms Mus musculus, Homo sapiens
Diseases Lupus Erythematosus, Systemic
Chemicals Adenosine Triphosphate, Guanine