For in vivo treatment with TLR ligands, mice were anesthetized and injected i.v. in the retro-orbital vein with 200 μl of PBS (Life Technologies), LPS (5 μg in 200 μl of PBS, Sigma-Aldrich), resiquimod (R-848, 5 μg in 200 μl of PBS; Invitrogen), or CpG-ODN preparation. This preparation consisted of 5 μg of CpG-ODN (TCA TTG GAA AAC GTT CTT CGG GGC G, phosphorothioate-modified, from MWG Biotech AG) mixed with 30 μl of a cationic liposome preparation (DOTAP; F. Hoffmann-La Roche Ltd.) and 170 μl PBS in a polystyrene tube. The preparation was injected using a glass syringe. MCMV infections were initiated on day zero by intraperitoneal injection of 5 × 104 plaque-forming units of salivary gland–extracted MCMV, Smith strain. For all in vivo treatment, at the indicated time point, mice were killed by CO2 inhalation, and spleens were collected for further analysis. If isolated spleen cells were needed, spleens were crushed in cold PBS supplemented with 5% (vol/vol) heat-inactivated FCS and 0.5 mM EDTA (Sigma-Aldrich) (PBS-FCS-EDTA) and passed through a 25-G needle. RBCs were lysed in NH4Cl solution (StemCell Technologies, Inc.) for 5 min at 4°C. Spleen DCs were enriched from total spleen cells by positive selection using CD11c+ Microbeads and MiniMacs (Miltenyi Biotec).
For analysis of in vivo cytokine and chemokine production, blood was collected by cardiac puncture at the indicated time point after treatment. Serum was prepared from whole blood by coagulation for 30 min at 37°C and centrifugation. Sera were titrated for mouse IFN-α using specific ELISA (PBL Biomedical Laboratories) and for mouse natural type I IFN using a conventional cytopathic effect inhibition bioassay on L-929 cells (CCL-1; American Type Culture Collection), infected with Encephalomyocarditis virus (EMCV, VR-129B, ATCC). All biological assays were performed in the presence of a neutralizing rat anti–mouse IFN-γ mAb (clone R4-6A2), produced in the laboratory. When needed, the assay was performed in the presence of a mixture of neutralizing rabbit anti-IFNα and IFNβ pAb (PBL Biomedical Laboratories). CXCL9, CXCL10, and CCL21 were assayed using specific ELISA (R&D Systems).
For analysis of in vivo cytokine and chemokine production, blood was collected by cardiac puncture at the indicated time point after treatment. Serum was prepared from whole blood by coagulation for 30 min at 37°C and centrifugation. Sera were titrated for mouse IFN-α using specific ELISA (PBL Biomedical Laboratories) and for mouse natural type I IFN using a conventional cytopathic effect inhibition bioassay on L-929 cells (CCL-1; American Type Culture Collection), infected with Encephalomyocarditis virus (EMCV, VR-129B, ATCC). All biological assays were performed in the presence of a neutralizing rat anti–mouse IFN-γ mAb (clone R4-6A2), produced in the laboratory. When needed, the assay was performed in the presence of a mixture of neutralizing rabbit anti-IFNα and IFNβ pAb (PBL Biomedical Laboratories). CXCL9, CXCL10, and CCL21 were assayed using specific ELISA (R&D Systems).
