αcd86 fitc

Extracellular surface marker staining was performed with IgG1-FITC, IgG2a-FITC, IgG1-PE, αCD25-FITC, αCD40-PE, αCD69-PE, αCD70-PE, αCD80-FITC, αCD83-PE, αCD86-FITC, and PD-L1-PE (all from BD Biosciences, Heidelberg, Germany); IgG3-PE (eBioscience, Frankfurt, Germany); and αCCR7-FITC (R&D Systems, Minneapolis, MN, USA) for 30 min at 4 °C in PBS supplemented with 1% FCS and 0.02% sodium azide (Merck, Darmstadt, Germany). The cells were analyzed using a FACScan cytofluorometer equipped with CellQuest software (BD, Heidelberg, Germany). Analysis was performed with the FCS Express software (De Novo Software, Glendale, CA, USA). Specific MFIs were calculated by subtracting the background MFI obtained with the isotype controls.

Femurs were surgically excised from wt and CD200R^-/- mice, sterilized in 70% ethanol and washed in PBS. Bone marrow was isolated, cells centrifuged, washed in RPMI and passed through a 40µM cell strainer. Cells were incubated at 2 x 10⁵ cells/well in D10 media supplemented with 20ng/ml of M-CSF (Peprotech) for 7 days, replenishing M-CSF after 3 days. Spleens and SGs were processed as previously described, with an additional Percoll (GE Healthcare) purification step for SGs after processing. Bone-marrow derived macrophages were infected with MCMV or influenza (PR8) at an MOI of 1. Some cells were also incubated with 300μg/ml phosphonoacetic acid (PAA, Sigma-Aldrich) for 1 hour prior to infection. Splenocytes (2 x 10⁵ cells/well) and SG leukocytes (2 x 10⁴ cells/well) were infected in 48-well plates and infected with MOI 0.5 MCMV. After 24hrs, all macrophages were gently scraped gently off the bottom of the wells, stained with Live/Dead® fixable aqua dead cell stain (Invitrogen) and Fc block (eBioscience), and surface stained with αCD200-PE (Clone OX-90, Biolegend), αCD80 Pacific blue (Clone 16–10A1, Biolegend), αCD86 FITC (Clone GL-1, BD Pharmingen), and αIA/IE PerCP/Cy5.5 (Clone M5/114.15.2, Biolegend) prior to permeabilization and staining with anti-m06 antibody (a kind gift from Stipan Jonjic, Rijeka) conjugated with APC (Innova Biosciences).