Murine ifn β

THP-1 cells were infected with VSV (MOI = 0.05) for 16 h. The supernatant was collected and used to determine cytokine concentration.
Mice were infected with HSV-1 or VSV and sera were collected at 8 h post-infection for cytokine detection.
ELISA kit was used to determine the concentration of human IFN-β (PBL Assay Science), murine IFN-β (PBL Assay Science), CCL5 (R&D systems), and IL-6 (BD Biosciences) according to the manufacturer’s instructions.

For cell experiments, cells were inoculated with HMPV strain TN/94-49 at an MOI of 1–10 in a 24-well plate. Mock-infected cells were inoculated with media or LLC-MK2 cell lysate, which had an equivalent effect on STAT1 and STAT2 protein levels and phosphorylation [30 ]. Then, 16–24 h post-infection, cells were treated with 1000 U/mL human IFNα (Alpha 2a) (PBL; Piscataway, NJ, USA) for human and primate cells, or 1000 U/mL murine IFNβ (PBL) for mouse cells for 30–40 min. After treatment, media were aspirated from the tissue culture dish and cells were lysed in RIPA buffer (ThermoFisher; Waltham, MA, USA) for western blotting or fixed in 4% paraformaldehyde for immunofluorescence.

Peritoneal cells were isolated from naïve mice by lavage using 10 mL of ice cold PBS from the peritoneal cavity. Non-tissue culture treated suspension plates were used to minimize cell adherence for experiments involving flow cytometric surface expression analysis. For experiments using western blot analysis, peritoneal cells were placed on tissue-culture treated plates for several hours to ensure adherence by the macrophages and non-adherent cells were removed by vigorous washes with PBS. Cells were cultured in DM10 media (DMEM supplemented with 10% FBS, 1% sodium pyruvate, 1% L-glutamine, 1% penicillin/streptomycin). In experiments evaluating IFNGR1, cells were treated with 100 Units (U)/mL murine IFNβ (PBL, #12401–1) for 6–8 hrs. For experiments evaluating MHC II up regulation cells were treated ± 100 U/mL IFNβ for 6–8 hrs followed by treatment with 100 U/mL murine IFNγ (LifeTechnology, #PMC-4031) for 18–24 hrs. For MHC I expression, cells were treated with 100 U/mL IFNβ for 24 hrs.

Monolayers of tumor cells were treated with 250 U/mL of murine IFNβ (PBL interferon source, Piscataway, NJ, USA) 4 h prior to virus infection. The production of IFNβ by tumor cells was quantified using the ELISA mouse IFNβ kit (R&D systems, Minneapolis, MN, USA) following the manufacturer’s protocol. The samples were generated by pre-treating the cells with PAC as described above and infecting them for 24 h at an MOI of 0.1.