Isolation of peritoneal macrophages and challenge with PVM

Peritoneal cavities of naïve mice were lavaged with RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin and 25 μM 2-mercaptoethanol. Peritoneal fluids were pooled and centrifuged (400 × g, 5 min). Pelleted cells were suspended in DMEM/F12 supplemented with 10% FBS, 2 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin and 100 IU/mL M-CSF and seeded in culture plates. Cells were permitted to adhere to the plates for 40 min prior to washing with PBS. Routinely, more than 90% of the cells obtained by this procedure displayed a macrophage phenotype as assessed by visual inspection and by anti-F4/80 staining. Peritoneal macrophages were challenged 24 hours later with PVM, heat-inactivated PVM (_hiPVM; PVM virions heated to 95°C and then flash frozen in a dry ice–methanol bath, repeated 3 times) at a multiplicity of infection (MOI) of 100 or were sham treated with an equal volume of IMDM. The cells and virus were co-incubated for 4 hours at 37°C in a humidified 5% CO₂ incubator, after which the cells were washed twice with PBS and placed in fresh complete medium. At the time points indicated, aliquots of culture media were collected for analysis of virus recovery by qRT-PCR, cytokine release by ELISA (R&D Systems), or Western blot probed with anti-PVM N antibody (Percopo et al., 2011) and anti-beta actin to determine relative loading (Cell Signaling, Millipore Corporation).