Streptavidin apc

ELISAs specific for murine soluble IL-15Rα/IL-15 complexes (eBioscience, San Diego, CA) and murine IFN-α (detects all 14 IFN-α subtypes, PBL Biomedical Laboratories) were performed according to manufacturer’s recommendations. Cell surface IL-15 was detected in splenic myeloid cells isolated directly ex vivo as previously described [29 (link)]. Briefly, cell surface IL-15 was detected with polyclonal rabbit anti-IL-15-biotin (Peprotech, Rocky Hill, NJ) followed by streptavidin-APC (Jackson ImmunoResearch). Background staining was determined by staining analogous populations with a biotinylated Ig control (Jackson ImmunoResearch). The following monoclonal (m) Abs were purchased from BD Biosciences (San Jose, CA), eBiosciences, or BioLegend: CD19, CD3, DX5, CD11b, CD11c. Expression of CD19, CD3 and DX5 was used to define lineage+ cells. Flow cytometric data were acquired with a LSRII (BD Biosciences) or LSRFortessa (BD Biosciences) and analyzed with Flowjo software version 9.7.6 (Flowjo LLC, Ashland, OR).

MRC-5 cells were infected with 1 PFU/cell of wt HCMV and HCMV ΔvFcγR mutants for 72 h and with 10 PFU/cell of HSV-1 wt and ΔgE for 24 h. Cells were resuspended in PBS containing 2 mM EDTA, washed twice in PBS supplemented with 3% (vol/vol) FCS. HCMV infected cells were stained with the F(ab)₂ preparation of Cytotect, goat anti-human-F(ab)₂-Biotin, and Streptavidin-PE (AdB Serotec, UK) or Fcγ fragment-FITC (Rockland Immunochemicals, USA). The comparability of infection of the different HCMV ΔvFcγR mutants was controlled by intracellular staining of CMV nuclear antigens with CCH2 and DDG9 antibodies (Dako, Denmark) and goat anti-mouse-APC (BD Pharmingen, USA) after fixation with 1,5% PFA and permeabilization with PBS supplemented with 3% (vol/vol) FCS and 0,05% (vol/vol) Saponin. HSV infected cells were stained with the F(ab)₂ preparation of Cytotect, goat anti-human-F(ab)₂-Biotin (AdB Serotec, UK) or Fcγ fragment-Biotin (Rockland Immunochemicals, USA) and Streptavidin-APC (Jackson Immunoresearch, USA). After DAPI staining, 1–2×10⁴ living cells were obtained in a FACSCanto II using the FACS Diva software and analyzed with FlowJo (Tree Star Inc, USA).